UNITED NATIONS ENVIRONMENT PROGRAMME
INTERNATIONAL LABOUR ORGANISATION
WORLD HEALTH ORGANIZATION
INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY
Basic Analytical Toxicology
The issue of this document does not constitute formal publication.
It should not be reviewed, abstracted, or quoted without the written
permission of the Manager, International Programme on Chemical Safety,
WHO, Geneva, Switzerland.
This report contains the collective views of an international group of
experts and does not necessarily represent the decisions or the stated
policy of the United Nations Environment Programme, the International
Labour Organisation, or the World Health Organization.
Basic analytical toxicology
R.J. Flanagan
Guy's and St Thomas' Hospital NHS Trust
London, England
R.A. Braithwaite
Regional Laboratory for Toxicology
City Hospital NHS Trust
Birmingham, England
S.S. Brown
Formerly Regional Laboratory for Toxicology
City Hospital NHS Trust
Birmingham, England
B. Widdop
Guy's and St. Thomas' Hospital NHS Trust
London, England
F.A. de Wolff
Department of Human Toxicology, Academic Medical Centre
University of Amsterdam
Amsterdam, Netherlands
World Health Organization
Geneva, 1995
The International Programme on Chemical Safety (IPCS) is a joint
venture of the United Nations Environment Programme, the International
Labour Organisation, and the World Health Organization. The main
objective of the IPCS is to carry out and disseminate evaluations of
the effects of chemicals on human health and the quality of the
environment. Supporting activities include the development of
epidemiological, experimental laboratory, and risk-assessment methods
that could produce internationally comparable results, and the
development of manpower in the field of toxicology. Other activities
carried out by the IPCS include the development of know-how for coping
with chemical accidents, coordination of laboratory testing and
epidemiological studies, and promotion of research on the mechanisms
of the biological action of chemicals.
WHO Library Cataloguing in Publication Data
Basis analytical toxicology/R.J. Flanaga...[et al.].
1.Poisions 2.Poisons - analysis 3.Poisoning - laboratory manuals
I.Flanagan, R.J.
ISBN 92 4 15448 9 (NLM Classification: QV 602)
The World Health Organization welcomes requests for permission to
reproduce or translate its publications, in part or in full.
Applications and enquiries should be addressed to the Office of
Publications, World Health Organization, Geneva, Switzerland, which
will be glad to provide the latest information on any changes made to
the text, plans for new editions, and reprints and translations
already available.
(c) World Health Organization 1995
Publications of the World Health Organization enjoy copyright
protection in accordance with the provisions of Protocol 2 of the
Universal Copyright Convention. All rights reserved. The designations
employed and the presentation of the material in this publication do
not imply the expression of any opinion whatsoever on the part of the
Secretariat of the World Health Organization concerning the legal
status of any country, territory, city or area or of its authorities,
or concerning the delimitation of its frontiers or boundaries. The
mention of specific companies or of certain manufacturers' products
does not imply that they are endorsed or recommended by the World
Health Organization in preference to others of a similar nature that
are not mentioned. Errors and omissions excepted, the names of
proprietary products are distinguished by initial capital letters.
Contents
Preface
Acknowledgements
Introduction
1. Apparatus and reagents
1.1 Apparatus
1.2 Reference compounds and reagents
2. Clinical aspects of analytical toxicology
2.1 Diagnosis of acute poisoning
2.2 Treatment of acute poisoning
2.3 The role of the clinical toxicology laboratory
3. General laboratory findings in clinical toxicology
3.1 Biochemical tests
3.2 Haematological tests
4. Practical aspects of analytical toxicology
4.1 Laboratory management and practice
4.2 Colour tests
4.3 Pretreatment of samples
4.4 Thin-layer chromatography
4.5 Ultraviolet and visible spectrophotometry
5. Qualitative tests for poisons
5.1 Collection, storage and use of specimens
5.2 Analysis of urine, stomach contents and scene residues
6. Monographs - analytical and toxicological data
6.1 Amfetamine 6.2 Aminophenazone
6.3 Amitriptyline 6.4 Aniline
6.5 Antimony 6.6 Arsenic
6.7 Atenolol 6.8 Atropine
6.9 Barbiturates 6.10 Barium
6.11 Benzodiazepines 6.12 Bismuth
6.13 Borates 6.14 Bromates
6.15 Bromides 6.16 Cadmium
6.17 Caffeine 6.18 Camphor
6.19 Carbamate pesticides 6.20 Carbamazepine
6.21 Carbon disulfide 6.22 Carbon monoxide
6.23 Carbon tetrachloride 6.24 Chloral hydrate
6.25 Chloralose 6.26 Chlorates
6.27 Chloroform 6.28 Chlorophenoxy
herbicides
6.29 Chloroquine 6.30 Cholinesterase activity
6.31 Clomethiazole 6.32 Cocaine
6.33 Codeine 6.34 Copper
6.35 Coumarin 6.36 Cyanide
anticoagulants
6.37 Dapsone 6.38 Dextropropoxyphene
6.39 Dichloralphenazone 6.40 Dichloromethane
6.41 Digoxin and digitoxin 6.42 Dinitrophenol
pesticides
6.43 Diphenhydramine 6.44 Diquat
6.45 Ephedrine 6.46 Ethanol
6.47 Ethchlorvynol 6.48 Ethylene glycol
6.49 Fluoride 6.50 Fluoroacetate
6.51 Formaldehyde 6.52 Formic acid and formate
6.53 Glutethimide 6.54 Glyceryl trinitrate
6.55 Haloperidol 6.56 Hydroxybenzonitrile
herbicides
6.57 Hypochlorites 6.58 Imipramine
6.59 Iodates 6.60 Iodine and iodide
6.61 Iron 6.62 Isoniazid
6.63 Laxatives 6.64 Lead
6.65 Lidocaine 6.66 Lithium
6.67 Meprobamate 6.68 Mercury
6.69 Methadone 6.70 Methanol
6.71 Methaqualone 6.72 Methyl bromide
6.73 Morphine 6.74 Nicotine
6.75 Nitrates 6.76 Nitrites
6.77 Nitrobenzene 6.78 Nortriptyline
6.79 Organochlorine 6.80 Organophosphorus
pesticides pesticides
6.81 Orphenadrine 6.82 Oxalates
6.83 Paracetamol 6.84 Paraquat
6.85 Pentachlorophenol 6.86 Peroxides
6.87 Pethidine 6.88 Petroleum distillates
6.89 Phenacetin 6.90 Phenols
6.91 Phenothiazines 6.92 Phenytoin
6.93 Phosphorus and phosphides 6.94 Procainamide
6.95 Propan-2-ol 6.96 Propranolol
6.97 Propylene glycol 6.98 Quinine and quinidine
6.99 Salicylic acid and 6.100 Strychnine
derivatives
6.101 Sulfides 6.102 Sulfites
6.103 Tetrachloroethylene 6.104 Thallium
6.105 Theophylline 6.106 Thiocyanates
6.107 Tin 6.108 Tolbutamide
6.109 Toluene 6.110 1,1,1-Trichloroethane
6.111 Trichloroethylene 6.112 Verapamil
6.113 Zinc
Bibliography
Glossary
Annex 1. List of reference compounds and reagents
Annex 2. Conversion factors for mass and molar concentrations
Preface
For many years, toxicology, the science of poisons and poisoning,
was considered to be no more than a branch of forensic science and
criminology. Nowadays, it is clear that the study of applied
toxicology in its various forms - clinical, occupational, forensic,
nutritional, veterinary, and environmental toxicology, ecotoxicology
and related areas - is important, if not vital, to the continued
development of life on earth. Yet toxicology is rarely taught as a
subject in its own right and then mostly at postgraduate level. In
consequence, most toxicologists come to the subject under the auspices
of another discipline. Clinical toxicology, dealing with the
prevention, diagnosis and management of poisoning, is no exception,
being often thought of as a branch of emergency medicine and intensive
care on the one hand, and of clinical pharmacology on the other.
The provision of services for the management of poisoned patients
varies greatly, from specialized treatment units to, more commonly,
general emergency medicine. Analytical toxicology services, which
provide support for the diagnosis, prognosis and management of
poisoning, are also variable and dependent on local arrangements. In
developed countries, they may be provided by a specialized laboratory
attached to a clinical toxicology unit, by a hospital biochemistry
laboratory, an analytical pharmacy unit, a university department of
forensic medicine, or a government forensic science laboratory.
In many developing countries, such services are not available on a
regular basis, and where they are available, the physician is
generally dependent on a national or regional health laboratory
established for other purposes and operating only part of the time.
There are, however, many simple analytical techniques that do not need
sophisticated equipment or expensive reagents, or even a continuous
supply of electricity. Such tests could be carried out in the basic
laboratories that are available to most hospitals and health
facilities, even in developing countries. With training, hospital
laboratory staff could use these techniques to provide an analytical
toxicology service to the physicians treating poisoned patients.
This manual, which describes simple analytical techniques of this
kind, has been prepared on the recommendation of a group of experts,
convened by the International Programme on Chemical Safety (IPCS)a in
February 1987.
The draft text was reviewed by a number of experts, as noted under
"Acknowledgements", and the procedures described were tested in the
laboratory, as far as possible by technicians from developing
countries. The work was coordinated for IPCS by Dr J. Haines. The
United Kingdom Department of Health, through its financial support to
the IPCS, provided the resources for the editorial group to meet and
undertake its work.
The aim of this manual is to help hospital laboratories in
developing countries to provide a basic analytical toxicology service
using a minimum of special apparatus. It is not intended to replace
standard texts, but to provide practical information on the analysis
of a number of substances frequently involved in acute poisoning
incidents. Common pitfalls and problems are emphasized throughout, and
basic health and safety precautions for laboratory workers are also
discussed.
Problems encountered when using relatively simple methods in
analytical toxicology are usually due to interference (false
positives) or poor sensitivity (false negatives). Nevertheless, useful
information to help the clinician, and thus the patient, can often be
obtained if the tests are applied with due caution using an
appropriate sample. While every effort has been made to ensure that
the tests described are reliable and accurate, no responsibility can
be accepted by UNEP, ILO or WHO for the use made of the tests or of
the results obtained.
As in all areas of analytical chemistry, problems in
interpretation can arise if a result is used for purposes for which it
was not intended. This is especially true if the results of emergency
toxicological analyses, particularly if poorly defined (for example,
"negative drug screen", "opiates positive"), are used as evidence in
legal proceedings many months or even years later. In this context,
a The IPCS is a cooperative programme of the United Nations
Environment Programme (UNEP), the International Labour
Organisation (ILO) and the World Health Organization (WHO). WHO is
the executing agency for the programme, which aims to provide the
internationally evaluated scientific data basis for countries to
develop their own chemical safety measures and to strengthen
national capabilities to prevent and treat harmful effects of
chemicals and to manage chemical emergencies.
the importance of consultation between the clinician treating the
patient and the analyst in making best use of the analytical
facilities available cannot be over-emphasized. To assist this
dialogue, some information on clinical interpretation has been
included.
IPCS and the editorial group would welcome comments on the content
and structure of the manual; such comments should be addressed in the
first instance to the Director, International Programme on Chemical
Safety, World Health Organization, 1211 Geneva 27, Switzerland. Two
areas for further development have already been identified, namely,
the requirement for formal training in analytical toxicology, and the
need to ensure the supply of essential reference compounds,
specialized reagents and laboratory consumables. Comments on either of
these problems would also be welcome.
Acknowledgements
Many individuals have contributed to the preparation of this
manual by providing support, ideas, details of methods or comments on
various drafts. In particular, Professor Bahira Fahim, Cairo, Egypt,
Dr I. Sunshine, Palo Alto, CA, USA, and Dr G. Volans, London, England
provided initial encouragement. Dr T. J. Meredith, London, England,
Dr J. Pronczuk de Garbino, Montevideo, Uruguay, and Professor A. N. P.
van Heijst, Utrecht, Netherlands scrutinized the clinical information.
Dr A. Akintonwa, Lagos, Nigeria, Dr A. Badawy, Cairo, Egypt,
Dr N. Besbelli, Ankara, Turkey, Dr C. Heuck, WHO, Geneva, Switzerland,
Professor M. Geldmacher-von Mallinckrodt, Erlangen, Germany,
Mr R. Fysh, London, England, Professor R. Merad, Algiers, Algeria,
and Mr. J. Ramsey, Mr J. Slaughterr and Dr J. Taylor, London,
England kindly commented on various aspects of the final draft.
Miss H. Triador, Montevideo, Uruguay, Mrs K. Pumala, Bangkok, Thailand
and Mr J. Howard, London, England, undertook the onerous task of
critically evaluating many of the tests described. Finally, thanks are
due to Dr B. Abernethy and Mr D. Spender, Basingstoke, England for
help in preparing the text, and to Mr M. J. Lessiter, Birmingham,
England for help with the illustrations of spot tests and thin-layer
chromatography plates.
Introduction
After a brief introduction to the apparatus, reference compounds
and reagents needed for an analytical toxicology laboratory (section
1), the manual covers a number of general topics, namely, clinical
toxicology (section 2), clinical chemistry and haematology in relation
to clinical toxicology (section 3), practical aspects of analytical
toxicology (section 4), sample collection and storage, and qualitative
poisons screening (section 5). Then, in a series of monographs
(section 6), qualitative tests and some quantitative methods are
described for 113 specific poisons or groups of poisons. Each
monograph also includes some information on clinical interpretation.
The practical sections of the manual have been designed to be
followed at the bench so that full experimental details of a test for
a particular substance are often given, especially in the monographs
(section 6), even though these same details may be repeated elsewhere
in another context.
The tests described in sections 5 and 6 have been restricted to
those that can be expected to produce reliable results within the
limitations described, and that can be performed using relatively
simple apparatus. Where appropriate, tests applicable to powders,
tablets or other items found with or near the patient (scene residues)
and to biological fluids are also included. Additional simple tests
for specified pharmaceuticals are given in other World Health
Organization publications.a However, these are designed to test the
identity and in some cases stability of specific, relatively pure
compounds and little consideration is given to, for example,
purification procedures, sensitivity and sources of interference.
Primary references to particular methods have not been given, in
order to simplify presentation and also because many tests have been
modified over the years, so that reference back to the original paper
could cause confusion. However, much of the information given in the
manual can be found in the references listed in the Bibliography. An
attempt has been made to assess the sensitivity (detection limit) of
all the qualitative tests given in the monographs (section 6).
However, as with description of colour, such assessments are always to
some extent subjective. In addition, the sensitivity of some tests,
such as those involving solvent extraction, can usually be varied by
taking more (or less) sample. These points emphasize the importance of
analysing known negative (control) and positive (reference) samples
alongside every specimen (see section 4.1.5).
a Basic tests for pharmaceutical substances. Geneva, WHO, 1986;
Basic tests for pharmaceutical dosage forms. Geneva, WHO, 1991.
Many of the terms used in this manual are defined in the Glossary
and a list of reference compounds and reagents is provided (Annex 1).
Système internationale (International System; SI) mass units
(mg/l, µg/l, etc.) have been used throughout to express concentrations
of drugs and other poisons. There is a tendency to use SI molar units
(mmol/l, µmol/l, etc.) for this purpose, but this can cause
unnecessary confusion and has no clear advantage in analytical
toxicology, provided that the exact chemical form of a substance is
specified. SI mass/molar unit conversion factors for some common
poisons are given in Annex 2. In some cases, SI mass units have also
been used to express reagent concentrations, but it should be borne in
mind that it is often sensible to prepare quantities of reagent
smaller than one litre (100 ml, for example), especially for
infrequently used tests.
For convenience, trivial or common chemical names have been used
throughout the text; where necessary, IUPAC equivalents are given in
the index. International nonproprietary names are used in the text for
drugs; common synonyms are listed in the index.
1 Apparatus and reagents
1.1 Apparatus
Analytical toxicology services can be provided in clinical
biochemistry laboratories that serve a local hospital or accident
and emergency unit (of the type described in the WHO document
Laboratory services at the primary health care level).a In addition
to basic laboratory equipment, some specialized apparatus, such as
that for thin-layer chromatography, ultraviolet and visible
spectrophotometry and microdiffusion, is needed (Table 1). 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. For
example, 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, which will
usually require regular visits from the manufacturer's representative
or agent. Indeed, such visits may need to be more frequent in
developing countries, since the operating conditions (temperature,
humidity, dust) can be more severe than those encountered elsewhere.
a Unpublished document WHO/LAB/87.2. Available on request from
Health Laboratory Technology and Blood Safety, World Health
Organization, 1211 Geneva 27, Switzerland.
Table 1. Summary of basic equipment required for toxicological
analyses
Reliable, regularly serviced and calibrated laboratory balances
(top-pan and analytical) (section 4.1.3.)
Bench-top centrifuge (electrical or hand-driven) for separating
blood samples and solvent extracts (section 4.3.2)
Vortex-mixer or other form of mechanical or hand-driven shaker
such as a rotary mixer (section 4.3.2)
Water-bath and (electrical) heating block
Spirit lamp or butane gas burner
Refrigerator (electrical or evaporative) for storing
standards/samples
pH meter
Range of automatic and semi-automatic pipettes (section 4.1.3)
Low-power, polarizing microscope
An adequate supply of laboratory glassware, including volumetric
apparatus, and adequate cleaning facilities (section 4.1.5)
A supply of chemically pure water (section 4.1.4)
A supply of compressed air or nitrogen
A supply of thin-layer chromatography plates or facilities for
preparing such plates (section 4.4.1)
Facilities for developing and visualizing thin-layer
chromatograms, including an ultraviolet lamp (254 nm and
366 nm) and a fume cupboard or hood (section 4.4.4)
Single-beam or dual-beam ultraviolet/visible spectrophotometer
and associated cells (section 4.5.2)
Conway microdiffusion apparatus (section 4.3.3)
Porcelain spotting tile (section 4.2)
Modified Gutzeit apparatus (section 6.6)
Some drug-testing facilities are now available in kit form. For
example, there are standardized thin-layer chromatography (TLC) drug
screening systems, which have the advantage that the plates are dipped
or otherwise exposed to visualization reagents, and not sprayed, so
that a fume cupboard or hood (see section 4.4.4) is not required. In
addition, the interpretation of results is assisted by a compendium of
annotated colour photographs. However, as with conventional TLC
systems, interpretation can be difficult, especially if more than one
compound is present. Further, the availability of the system and its
associated consumables cannot be guaranteed.
Similarly, immunoassay kits are relatively simple to use,
although problems can arise in practice, especially in the
interpretation of results. Moreover, they are aimed primarily at the
therapeutic drug monitoring and drug abuse testing markets and, as
such, have limited direct application in clinical toxicology.
1.2 Reference compounds and reagents
A list of the reference compounds and reagents needed in a basic
analytical toxicology laboratory is given in Annex 1. A supply of
relatively pure compounds for use as reference standards is essential
if reliable results are to be obtained. However, expensive reference
compounds of a very high degree of purity, such as those marketed for
use as pharmaceutical quality control standards, are not normally
needed. Some drugs, such as barbital, caffeine and salicylic acid, and
many inorganic and organic chemicals and solvents are available as
laboratory reagents with an adequate degree of purity through normal
laboratory chemical suppliers. Small quantities of a number of
controlled drugs and some metabolites can be obtained from: Narcotics
Laboratory Section, United Nations Vienna International Centre, P.O.
Box 500, A-1400 Vienna, Austria.
It may be difficult to obtain small quantities (100 mg-1 g) of
other drugs, pesticides, and their metabolites in pure form.
Nevertheless, an attempt should be made to build up a reference
collection or library (see Annex 1) without waiting for individual
poisons to be found in patient samples. Such a reference collection is
a valuable resource, and it should be stored under conditions that
ensure safety, security and stability. If the pure compound cannot be
obtained, then a pharmaceutical or other formulation is often the next
best thing, and purification sufficient for at least a qualitative
analysis can often be achieved by solvent extraction followed by
recovery of the compound of interest (see section 4.1.2).
Although the apparatus required to perform the tests described in
this manual is relatively simple, several unusual laboratory reagents
are needed in order to be able to perform all of the tests described.
Whenever possible, the shelf-life (stability) of individual compounds
and reagents and any special precautions required in handling have
been indicated in the text.
2 Clinical aspects of analytical toxicology
The trained analytical toxicologist can play a useful role in the
management of patients poisoned with drugs or other chemicals.
However, optimal analytical performance is only possible when the
clinical aspects of the diagnosis and treatment of such patients are
understood. The analyst must therefore have a basic knowledge of
emergency medicine and intensive care, and must be able to communicate
with clinicians. In addition, a good understanding of pharmacology and
toxicology and some knowledge of active elimination procedures and the
use of antidotes are desirable. This chapter aims to provide some of
the basic information required.
2.1 Diagnosis of acute poisoning
2.1.1 Establishing a diagnosis
When acute poisoning is suspected, the clinician needs to ask a
number of questions in order to establish a diagnosis. In the case of
an unconscious (comatose) patient, the circumstances in which the
patient was found and whether any tablet bottles or other containers
(scene residues) were present can be important. If the patient is
awake, he or she should be questioned about the presence of poisons in
the home or workplace. The patient's past medical history (including
drugs prescribed and any psychiatric illness), occupation and hobbies
may also be relevant, since they may indicate possible access to
specific poisons.
Physical examination of the patient may indicate the poison or
class of poison involved. The clinical features associated with some
common poisons are listed in Table 2. For example, the combination of
pin-point pupils, hypersalivation, incontinence and respiratory
depression suggests poisoning with a cholinesterase inhibitor such as
an organophosphorus pesticide. However, the value of this approach is
limited if a number of poisons with different actions have been
absorbed. Moreover, many drugs have similar effects on the body, while
some clinical features may be the result of secondary effects such as
anoxia. Thus, if a patient is admitted with depressed respiration and
pin-point pupils, this strongly suggests poisoning with an opioid such
as dextropropoxyphene or morphine. However, if the pupils are dilated,
then other hypnotic drugs such as glutethimide may be present, or
cerebral damage may have occurred as a result of hypoxia secondary to
respiratory depression.
Diagnoses other than poisoning must also be considered. For
example, coma can be caused by a cerebrovascular accident or
uncontrolled diabetes as well as poisoning. The availability of the
results of urgent biochemical and haematological tests is obviously
important in these circumstances (see section 3). Finally, poisoning
with certain compounds may be misdiagnosed, especially if the patient
Table 2. Acute poisoning: clinical features associated with specific
poisons
Clinical feature Poison
Central nervous system
Ataxia Bromides, carbamazepine, ethanol, hypnotics/
sedatives, phenytoin, thallium
Coma Alcohols, hypnotics/sedatives, opioids,
tranquillizers, many other compounds
Convulsions Amitriptyline and other tricyclic
antidepressants, orphenadrine, strychnine,
theophylline
Respiratory tract
Respiratory depression Alcohols, hypnotics/sedatives, opioids,
tranquillizers, many other compounds
Pulmonary oedema Acetylsalicylic acid, chlorophenoxy
herbicides, irritant (non-cardiogenic)
gases, opioids, organic solvents, paraquat
Hyperpnoea Acetysalicylic acid, ethylene glycol,
hydroxybenzonitrile herbicides, isoniazid,
methanol, pentachlorophenol
Heart and circulation
Tachycardia Anticholinergics, sympathomimetics
Bradycardia Cholinergics, ß-blockers, digoxin, opioids
Hypertension Anticholinergics, sympathomimetics
Hypotension Ethanol, hypnotics/sedatives, opioids,
tranquillizers, many other compounds
Arrhythmias ß-Blockers, chloroquine, cyanide, digoxin,
phenothiazines, quinidine, theophylline,
tricyclic antidepressants
Eyes
Miosis Carbamate pesticides, opioids,
organophosphorus pesticides, phencyclidine,
phenothiazines
Mydriasis Amfetamine, atropine, cocaine, tricyclic
antidepressants
Nystagmus Carbamazepine, ethanol, phenytoin
Table 2. (Con't)
Clinical feature Poison
Body temperature
Hyperthermia Acetylsalicylic acid, dinitrophenol
pesticides, hydroxybenzonitrile herbicides,
pentachlorophenol, procainamide, quinidine
Hypothermia Carbon monoxide, ethanol, hypnotics/
sedatives, opioids, phenothiazines,
tricyclic antidepressants
Skin, hair and nails
Acne Bromides, organochlorine pesticides
Hair loss Thallium
Gastrointestinal tract
Hypersalivation Cholinesterase inhibitors, strychnine
Dry mouth Atropine, opioids, phenothiazines, tricyclic
antidepressants
Constipation Lead, opioids, thallium
Diarrhoea Arsenic, cholinesterase inhibitors, laxatives
Gastrointestinal Acetylsalicylic acid, caustic compounds
bleeding (strong acids/bases), coumarin
anticoagulants, indometacin
Liver damage Amanita toxins, carbon tetrachloride,
paracetamol, phosphorus (white)
Urogenital tract
Urinary retention Atropine, opioids, tricyclic antidepressants
Incontinence Carbamate pesticides, organophosphorus
pesticides
Kidney damage Amanita toxins, cadmium, carbon
tetrachloride, ethylene glycol, mercury,
paracetamol
presents in the later stages of the episode. Examples include:
cardiorespiratory arrest (cyanide), hepatitis (carbon tetrachloride,
paracetamol), diabetes (hypoglycaemics, including ethanol in young
children), paraesthesia (thallium), progressive pneumonitis (paraquat)
and renal failure (ethylene glycol).
2.1.2 Classification of coma
Loss of consciousness (coma) is common in acute poisoning,
especially if central nervous system (CNS) depressants are involved. A
simple system, the Edinburgh scale (see Table 3), is often used to
classify the depth or grade of coma of poisoned patients. This system
has the advantage that the severity of an episode can be easily
described in conversation with laboratory staff and with, for example,
poisons information services that may be consulted for advice.
Table 3. Classification of depth of coma using the Edinburgh Scale
Grade of coma Clinical features
1 Patient drowsy but responds to verbal commands
2 Patient unconscious but responds to minimal
stimuli (for example, shaking, shouting)
3 Patient unconscious and responds only to painful
stimuli (for example, rubbing the sternum)
4 Patient unconscious with no response to any
stimuli
2.2 Treatment of acute poisoning
2.2.1 General measures
When acute poisoning is suspected, essential symptomatic and
supportive measures are often taken before the diagnosis is confirmed.
If the poison has been inhaled, the patient 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 patients, 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). The absorption of any
residue remaining after gastric lavage can be minimized by leaving a
high dose of activated charcoal in the stomach. The role of gavage and
induced emesis in preventing absorption is currently being examined,
as is the effectiveness of a single dose of activated charcoal.
However, repeated oral administration of activated charcoal appears to
be effective in enhancing elimination of certain poisons. Oral
charcoal should not be given when oral administration of a
protective agent, such as methionine following paracetamol overdosage,
is contemplated.
Subsequently, most patients can be treated successfully using
supportive care alone. In severely poisoned patients, this may include
intravenous administration of anticonvulsants such as diazepam (see
benzodiazepines) or clomethiazole, or of antiarrhythmics such as
lidocaine, all of which may be detected if a toxicological analysis is
performed later. Lidocaine is also used as a topical anaesthetic and
is often found in urine as a result of incidental administration
during urinary tract catheterization. Drugs or other compounds may
also be given during investigative procedures such as lumbar puncture.
Specific therapeutic procedures, such as antidotal and active
elimination therapy are sometimes indicated. The results of either a
qualitative or a quantitative toxicological analysis may be required
before some treatments are commenced because they are not without risk
to the patient. In general, specific therapy is only started when the
nature and/or the amount of the poison(s) involved are known.
2.2.2 Antidotes/protective agents
Antidotes or protective agents are only available for a limited
number of poisons (see Table 4). Controversy surrounds the use of some
antidotes, such as those used to treat cyanide poisoning, while others
are themselves potentially toxic and should be used with care. Lack of
response to a particular antidote does not necessarily indicate the
absence of a particular type of poison. For example, the opioid
antagonist naloxone will rapidly and completely reverse coma due to
opioids such as morphine and codeine without risk to the patient,
except that an acute withdrawal response may be precipitated in
dependent subjects. However, a lack of response does not always mean
that opioids are not present, since another, non-opioid, drug may be
the cause of coma, too little naloxone may have been given, or hypoxic
brain damage may have followed a cardiorespiratory or respiratory
arrest.
2.2.3 Active elimination therapy
There are four main methods of enhancing elimination of the
poison from the systemic circulation: repeated oral activated
charcoal; forced diuresis with alteration of urine pH; peritoneal
dialysis and haemodialysis; and haemoperfusion.
Table 4. Some antidotes and protective agents used to treat acute
poisoninga
Antidote/agent Indication
Acetylcysteine Paracetamol
Atropine Carbamate pesticides, organophosphorus
pesticides
Deferoxamine Aluminium, iron
DMSAb Antimony, arsenic, bismuth, cadmium,
lead, mercury
DMPSc Copper, lead, mercury (elemental and
inorganic)
Ethanol Ethylene glycol, methanol
Antigen binding (Fab) Digoxin
antibody fragments
Flumazenil Benzodiazepines
Methionine Paracetamol
Methylene blue Oxidizing agents (chlorates, nitrites,
etc.)
Naloxone Opioids (codeine, pethidine, morphine,
etc.)
Obidoxime chloride Organophosphorus pesticides
(or pralidoxime iodide) (contraindicated with carbamate
pesticides)
Oxygen Carbon monoxide, cyanide
Physostigmine Atropine
Phytomenadione Coumarin anticoagulants, indanedione
(vitamin K1) anticoagulants
Potassium Theophylline, barium
Protamine sulfate Heparin
Prussian blued Thallium
Pyridoxine (vitamin B6) Isoniazid
Sodium calcium edetate Lead, zinc
a Information on specific antidotes is given in the IPCS/CEC
Evaluation of Antidotes Series; see Bibliography.
b Dimercaptosuccinic acid
c Dimercaptopropanesulfonate
d Potassium ferrihexacyanoferrate
The systemic clearance of compounds such as barbiturates,
carbamazepine, quinine and theophylline (and possibly also salicylic
acid and its derivatives) can be enhanced by giving oral activated
charcoal at intervals of 4-6 hours until clinical recovery is
apparent. To reduce transit time and thus reabsorption of the poison,
the charcoal is often given together with a laxative. This procedure
has the advantage of being totally noninvasive but is less effective
if the patient has a paralytic ileus resulting from the ingestion of,
for example, phenobarbital. Care must also be taken to avoid pulmonary
aspiration in patients without a gag reflex or in those with a
depressed level of consciousness.
The aim of forced diuresis is to enhance urinary excretion of the
poison by increasing urine volume per unit of time. It is achieved by
means of intravenous administration of a compatible fluid. Nowadays,
forced diuresis is almost always combined with manipulation of urine
pH. Renal elimination of weak acids such as chlorophenoxy herbicides
and salicylates can be increased by the intravenous administration of
sodium bicarbonate. This can also protect against systemic toxicity
by favouring partition into aqueous compartments such as blood.
Indeed, alkalinization alone can be as effective as traditional
forced alkaline diuresis, and has the advantage that the risk of
complications resulting from fluid overload, such as cerebral or
pulmonary oedema and electrolyte disturbance, is minimized. However,
the pK of the poison must be such that renal elimination can be
enhanced by alterations in urinary pH within the physiological range.
It is also important to monitor urine pH carefully to ensure that the
desired change has been achieved. Acidification of urine was thought
to enhance the clearance of weak bases such as amfetamine,
procyclidine and quinine, but this is no longer generally accepted.
Dialysis and haemoperfusion remove the poison directly from the
circulation. In haemodialysis, blood is passed over a membrane which
is in contact with the aqueous compartment in an artificial kidney,
while in peritoneal dialysis an appropriate fluid is infused into the
peritoneal cavity and then drained some 2-4 hours later. In
haemoperfusion, blood is pumped through a cartridge of adsorbent
material (coated activated charcoal or Amberlite XAD-4 resin).
Haemodialysis is preferred for water-soluble substances such as
ethanol, and haemoperfusion for lipophilic poisons such as short-
acting barbiturates, which have a high affinity for coated charcoal or
Amberlite XAD-4 resin. The decision to use dialysis or haemoperfusion
should be based on the clinical condition of the patient, the
properties of the poison ingested and its concentration in plasma.
Haemodialysis and haemoperfusion are only effective when the volume of
distribution of the poison is small, i.e., relative volume of
distribution less than 5 l/kg.
2.3 The role of the clinical toxicology laboratory
Most poisoned patients can be treated successfully without any
contribution from the laboratory other than routine clinical
biochemistry and haematology. This is particularly true for those
cases where there is no doubt about the poison involved and when the
results of a quantitative analysis would not affect therapy. However,
toxicological analyses can play a useful role if the diagnosis is in
doubt, the administration of antidotes or protective agents is
contemplated, or the use of active elimination therapy is being
considered. The analyst's dealings with a case of poisoning are
usually divided into pre-analytical, analytical and post-analytical
phases (see Table 5).
Table 5. Steps in undertaking an analytical toxicological
investigation
Step Action
Pre-analytical phase
1. Obtain details of current admission,
including any circumstantial evidence of
poisoning and results of biochemical and
haematological investigations (see section 3).
2. Obtain patient's medical history, if
available, ensure access to the
appropriate sample(s), and decide
the priorities for the analysis.
Analytical phase
3. Perform the agreed analyses.
Post-analytical phase
4. Interpret the results and discuss them with
the clinician looking after the patient.
5. Perform additional analyses, if indicated,
on the original samples or on further
samples from the patient.
Practical aspects of the collection, transport, and storage of
the samples appropriate to a particular analysis are given in section
5 and in the monographs (section 6). Tests for any poisons that the
patient is thought to have taken and for which specific therapy is
available will normally be given priority over coma screening. This
topic is discussed fully in section 5 where a poisons screen is also
outlined. Tests for individual poisons or groups of poisons are given
in section 6.
Finally, an attempt must always be made to correlate the
laboratory findings with clinical observations. In order to do so,
some knowledge of the toxicological effects of the substances in
question is required (see Table 2). Additional information on
individual poisons is given in the monographs (section 6) and in the
clinical toxicology textbooks listed in the Bibliography. Some
instances where treatment might be influenced by the results of
toxicological analyses are listed in Table 6.
Table 6. Interpretation of emergency toxicological analyses
Poison Concentrationa Treatment
associated with
serious toxicity
1. Protective therapy
Paracetamol 200 mg/l at 4 h after )
ingestion ) Acetylcysteine
30 mg/l at 15 h after ) or methionine
ingestion )
Methanol 0.5 g/l )
Ethylene glycol 0.5 g/l ) Ethanol
Thallium 0.2 mg/l (urine) Prussian blueb
2. Chelation therapy
Iron 5 mg/l (serum) )
Aluminium 50-250 µg/l (serum) ) Deferoxamine
Lead 1 mg/l (whole blood, DMSAc/DMPSd/
adults) Sodium calcium edetate
Cadmium 20 µg/l (whole blood) DMSA
Mercury 100 µg/l (whole blood) DMSA/DMPS
Arsenic 200 µg/l (whole blood) DMSA
3. Active elimination therapy
Acetylsalicylic acid 900 mg/l at 6 hours after )
(as salicylate) ingestion )
450 mg/l at 24 hours after )
ingestion ) Alkaline diuresis
Phenobarbital 200 mg/l )
Barbital 300 mg/l )
Chlorophenoxy 500 mg/l )
herbicides
Ethanol 5 g/l )
Methanol 0.5 g/l )
Ethylene glycol 0.5 g/l )
Phenobarbital 200 mg/l ) Peritoneal dialysis
Barbital 300 mg/l ) or haemodialysis
Acetylsalicylic acid 900 mg/l at 6 h, )
(as salicylate) 450 mg/l at 24 h )
Lithium 14 mg/l )
Table 6. (Con't)
Poison Concentrationa Treatment
associated with
serious toxicity
Phenobarbital 100 mg/l )
Barbital 200 mg/l ) Charcoal
Other barbiturates 50 mg/l ) haemoperfusion
Theophylline 100 mg/l )
a In plasma, unless otherwise specified.
b Potassium ferrihexacyanoferrate
c Dimercaptosuccinic acid
d Dimercaptopropanesulfonate
3 General laboratory findings in clinical toxicology
Many clinical laboratory tests can be helpful in the diagnosis of
acute poisoning and in assessing prognosis. Those discussed here
(which are listed in Table 7) are likely to be the most useful,
although only the largest laboratories may be able to offer all of
them on an emergency basis. More specialized tests may be appropriate
depending on the clinical condition of the patient, the circumstantial
evidence of poisoning and the past medical history. Tests used in
monitoring supportive treatment are not considered here; details of
such tests can be found in standard clinical chemistry textbooks (see
Bibliography).
3.1 Biochemical tests
3.1.1 Blood glucose
Marked hypoglycaemia often results from overdosage with insulin,
sulfonylureas, such as tolbutamide, or other antidiabetic drugs.
Hypoglycaemia may also complicate severe poisoning with a number of
agents including iron salts and certain fungi, and may follow
ingestion of acetylsalicylic acid, ethanol (especially in children or
fasting adults) and paracetamol if liver failure ensues. Hypoglycin is
a potent hypoglycaemic agent found in unripe ackee fruit (Blighia
sapida) and is responsible for Jamaican vomiting sickness.
Hyperglycaemia is a less common complication of poisoning than
hypoglycaemia, but has been reported after overdosage with
acetylsalicylic acid, salbutamol and theophylline.
3.1.2 Electrolytes, blood gases and pH
Coma resulting from overdosage with hypnotic, sedative,
neuroleptic or opioid drugs is often characterized by hypoxia and
respiratory acidosis. Unless appropriate treatment is instituted,
however, a mixed acid-base disturbance with metabolic acidosis will
supervene. In contrast, overdosage with salicylates such as
acetylsalicylic acid initially causes hyperventilation and respiratory
alkalosis, which may progress to the mixed metabolic acidosis and
hypokalaemia characteristic of severe poisoning. Hypokalaemia and
metabolic acidosis are also features of theophylline and salbutamol
overdosage. Hypokalaemia occurs in acute barium poisoning, but severe
acute overdosage with digoxin gives rise to hyperkalaemia.
Toxic substances or their metabolites, which inhibit key steps in
intermediary metabolism, are likely to cause metabolic acidosis owing
to the accumulation of organic acids, notably lactate. In severe
poisoning of this nature, the onset of metabolic acidosis can be rapid
and prompt corrective treatment is vital. Measurement of the serum or
plasma anion gap can be helpful in distinguishing toxic metabolic
acidosis from that associated with nontoxic faecal or renal loss of
Table 7. Some laboratory tests likely to be useful in clinical
toxicology
Fluid Qualitative test Quantitative test
Urine Colour (haematuria, Relative density
myoglobinuria pH
Smell
Turbidity
Crystalluria
Blood Colour pCO2, pO2, pH
(oxygenation) Glucose
Prothrombin time
Carboxyhaemoglobin
Methaemoglobin
Erythrocyte volume fraction
(haematocrit)
Leukocyte count
Platelet count
Plasma Lipaemia Bilirubin
Electrolytes (Na+, K+, Ca2+,
Cl-, HCO3-)
Lactate
Osmolality
Plasma enzymesa
Cholinesterase
a Lactate dehydrogenase, aspartate aminotransferase, alanine
aminotransferase, creatine kinase
bicarbonate. The anion gap is usually calculated as the difference
between the sodium concentration and the sum of the chloride and
bicarbonate concentrations. It is normally about 10 mmol/l and also
corresponds to the sum of plasma potassium, calcium and magnesium
concentrations. This value is little changed in nontoxic metabolic
acidosis. However, in metabolic acidosis resulting from severe
poisoning with carbon monoxide, cyanide, ethylene glycol, methanol,
fluoroacetates, paraldehyde or acetylsalicylic acid, the anion gap may
exceed 15 mmol/l. Toxic metabolic acidosis may also occur in severe
poisoning with iron, ethanol, paracetamol, isoniazid, phenformin and
theophylline.
Other acid-base or electrolyte disturbances occur in many types
of poisoning for a variety of reasons. Such disturbances are sometimes
simple to monitor and to interpret, but are more often complex. The
correct interpretation of serial measurements requires a detailed
knowledge of the therapy administered. Hyperkalaemia or hypernatraemia
occurs in iatrogenic, accidental or deliberate overdosage with
potassium or sodium salts. The consequences of electrolyte imbalances
depend on many factors, including the state of hydration, the
integrity of renal function, and concomitant changes in sodium,
calcium, magnesium, chloride and phosphate metabolism. Hyponatraemia
can result from many causes, including water intoxication,
inappropriate loss of sodium, or impaired excretion of water by the
kidney. Hypocalcaemia can occur in ethylene glycol poisoning owing to
sequestration of calcium by oxalic acid.
3.1.3 Plasma osmolality
The normal osmolality of plasma (280-295 mOsm/kg) is largely
accounted for by sodium, urea and glucose. Unusually high values
(>310 mOsm/kg) can occur in pathological conditions such as gross
proteinaemia or severe dehydration where the effective proportion of
water in plasma is reduced. However, large increases in plasma
osmolality may follow the absorption of osmotically active poisons
(especially methanol, ethanol or propan-2-ol) in relatively large
amounts. Ethylene glycol, acetone and some other organic substances
with a low relative molecular mass are also osmotically active in
proportion to their molar concentration (see Table 8).
Table 8. Effect of some common poisons on plasma osmolality
Compound Plasma osmolality Analyte concentration
increase (mOsm/kg) (g/l) corresponding to
for 0.01 g/l 1 mOsm/kg increase
in plasma osmolality
Acetone 0.18 0.055
Ethanol 0.22 0.046
Ethylene glycol 0.20 0.050
Methanol 0.34 0.029
Propan-2-ol 0.17 0.059
Although the measurement of plasma osmolality can give useful
information, interpretation can be difficult. For example, there may
be secondary dehydration, as in overdosage with salicylates, ethanol
may have been taken together with a more toxic, osmotically active
substance, or enteral or parenteral therapy may have involved the
administration of large amounts of sugar alcohols (mannitol, sorbitol)
or formulations containing glycerol or propylene glycol.
3.1.4 Plasma enzymes
Shock, coma, and convulsions are often associated with
nonspecific increases in the plasma or serum activities of enzymes
(lactate dehydrogenase, aspartate aminotransferase, alanine
aminotransferase) commonly measured to detect damage to the major
organs. Usually the activities increase over a period of a few days
and slowly return to normal values. Such changes are of little
diagnostic or prognostic value.
The plasma activities of liver enzymes may increase rapidly after
absorption of toxic doses of substances that can cause liver necrosis,
notably paracetamol, carbon tetrachloride, and copper salts. It may
take several weeks for values to return to normal. The plasma
activities of the aminotransferases may be higher than normal in
patients on chronic therapy with drugs such as valproic acid, and
serious hepatotoxicity may develop in a small proportion of patients.
Chronic ethanol abuse is usually associated with increased plasma
gamma-glutamyltransferase activity.
In very severe poisoning, especially if a prolonged period of
coma, convulsions or shock has occurred, there is likely to be
clinical or subclinical muscle injury associated with rhabdomyolysis
and disseminated intravascular coagulation. Such damage can also occur
as a result of chronic parenteral abuse of psychotropic drugs. Frank
rhabdomyolysis is characterized by high serum aldolase or creatine
kinase activities together with myoglobinuria. This can be detected by
o-toluidine-based reagents or test strips, provided there is no
haematuria. In serious cases of poisoning, for example with
strychnine, myoglobinuria together with high serum or plasma
potassium, uric acid and phosphate concentrations may indicate the
onset of acute kidney failure.
3.1.5 Cholinesterase activity
Systemic toxicity from carbamate and organophosphorus pesticides
is due largely to the inhibition of acetylcholinesterase at nerve
synapses. Cholinesterase, derived initially from the liver, is also
present in plasma, but inhibition of plasma cholinesterase is not
thought to be physiologically important. It should be emphasized that
cholinesterase and acetylcholinesterase are different enzymes: plasma
cholinesterase can be almost completely inhibited while erythrocyte
acetylcholinesterase still possesses 50% activity. This relative
inhibition varies between compounds and with the route of absorption
and depending on whether exposure has been acute, chronic or acute-on-
chronic. In addition, the rate at which cholinesterase inhibition is
reversed depends on whether the inhibition was caused by carbamate or
organophosphorus pesticides.
In practice, 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 difficulty lies in deciding whether a low
activity is indeed due to poisoning or to some other physiological,
pharmacological or genetic cause. The diagnosis can sometimes be
assisted by detection of a poison or metabolite in a body fluid, but
the simple methods available are relatively insensitive (see sections
6.19 and 6.80). Alternatively pralidoxime, used as an antidote in
poisoning with organophosphorus pesticides (see Table 4), can be added
to a portion of the test plasma or serum in vitro (section 6.30).
Pralidoxime antagonises the effect of organophosphorus compounds on
cholinesterase. Therefore if cholinesterase activity is maintained in
the pralidoxime-treated portion of the sample but inhibited in the
portion not treated with pralidoxime, this provides strong evidence
that an organophosphorus compound is present.
Erythrocyte (red cell) acetylcholinesterase activity can be
measured, but this enzyme is membrane-bound and the apparent activity
depends on the methods used in solubilization and separation from
residual plasma cholinesterase. At present there is no standard
procedure. Erythrocyte acetylcholinesterase activity also depends on
the rate of erythropoiesis. Newly formed erythrocytes have a high
activity which diminishes with time. Hence erythrocyte
acetylcholinesterase activity is a function of the number and age of
the cell population. However, low activities of both plasma
cholinesterase and erythrocyte acetylcholinesterase is strongly
suggestive of poisoning with either organophosphorus or carbamate
pesticides.
3.2 Haematological tests
3.2.1 Blood clotting
Prolonged prothrombin time is a valuable early indicator of liver
damage in poisoning with metabolic toxins such as paracetamol. The
prothrombin time and other measures of blood clotting are likely to be
abnormal in acute poisoning with rodenticides such as coumarin
anticoagulants, and after overdosage with heparin or other
anticoagulants. Coagulopathies may also occur as a side-effect of
antibiotic therapy. The occurrence of disseminated intravascular
coagulation together with rhabdomyolysis in severe poisoning cases
(prolonged coma, convulsions, shock) has already been discussed
(section 3.1.4).
3.2.2 Carboxyhaemoglobin and methaemoglobin
Measurement of blood carboxyhaemoglobin can be used to assess
the severity of acute carbon monoxide poisoning and chronic
dichloromethane poisoning. However, carboxyhaemoglobin is dissociated
rapidly once the patient is removed from the contaminated atmosphere,
especially if oxygen is administered, and the sample should therefore
be obtained as soon as possible after admission. Even then, blood
carboxyhaemoglobin concentrations tend to correlate poorly with
clinical features of toxicity.
Methaemoglobin (oxidized haemoglobin) may be formed after
overdosage with dapsone and oxidizing agents such as chlorates or
nitrites, and can be induced by exposure to aromatic nitro compounds
(such as nitrobenzene and aniline and some of its derivatives). The
production of methaemoglobinaemia with intravenous sodium nitrite
is a classical method of treating acute cyanide poisoning.
Methaemoglobinaemia may be indicated by the presence of dark
chocolate-coloured blood. Blood methaemoglobin can be measured but is
unstable and results from stored samples are unreliable.
3.2.3 Erythrocyte volume fraction (haematocrit)
Acute or acute-on-chronic overdosage with iron salts,
acetylsalicylic acid, indometacin, and other nonsteroidal anti-
inflammatory drugs may cause gastrointestinal bleeding leading to
anaemia. Anaemia may also result from chronic exposure to toxins that
interfere with haem synthesis, such as lead, or induce haemolysis
either directly (arsine, see arsenic) or indirectly because of
glucose-6-phosphate dehydrogenase deficiency (chloroquine, primaquine,
chloramphenicol, nitridazole, nitrofurantoin).
3.2.4 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.
4 Practical aspects of analytical toxicology
It has been assumed that users of this manual will have some
practical knowledge of clinical chemistry and be familiar with basic
laboratory operations, including aspects of laboratory health and
safety. However, some aspects of laboratory practice are particularly
important if results are to be reliable and these are discussed in
this section.
Many of the topics discussed here and in sections 5 and 6 (use of
clinical specimens, samples and standards, pretreatment of samples,
thin-layer chromatography, ultraviolet/visible spectrophotometry) are
the subject of monographs in the Analytical Chemistry by Open Learning
(ACOL) series. The material contained in those monographs is
complementary to that given here, and the volumes will be useful to
those without a background in analytical chemistry. Details of ACOL
texts are given in the Bibliography.
4.1 Laboratory management and practice
4.1.1 Health and safety in the laboratory
Many of the tests described in this manual entail the use of
extremely toxic chemicals. The toxicity of some of them is not widely
recognized (the ingestion of as little as 20-30 ml of the commonly
used solvent methanol, for example, can cause serious toxicity in an
adult). Some specific hazards have been highlighted, but many have
been assumed to be self-evident - for example, strong acids and
alkalis should never be stored together, strong acids or alkalis
should always be added to water and not vice versa, organic solvents
should not be heated over a naked flame but in a water-bath, and a
fume cupboard or hood should always be used when organic solvents
are evaporated or thin-layer chromatography plates are sprayed with
visualization reagents.
Laboratory staff should be aware of local policies regarding
health and safety and especially of regulations regarding the
processing of potentially infective biological specimens. There should
also be a written health and safety policy that is available to, and
understood by, all staff, and there should be practical, written
instructions on how to handle and dispose of biological samples,
organic solvents and other hazardous or potentially hazardous
substances. A health and safety officer should be appointed from among
the senior laboratory staff with responsibility for the enforcement of
this policy. Ideally, disposable plastic gloves and safety spectacles
should be worn at all times in the laboratory. Details of the hazards
associated with the use of particular chemicals and reagents can often
be obtained from the supplier.
4.1.2 Reagents and drug standards
Chemicals obtained from a reputable supplier are normally graded
as to purity (analytical reagent grade, general purpose reagent,
laboratory reagent grade, etc.). The maximum limits of common or
important impurities are often stated on the label, together with
recommended storage conditions. Some chemicals readily absorb
atmospheric water vapour and either remain solid (hygroscopic, for
example the sodium salt of phenytoin) or enter solution (deliquescent,
for example trichloroacetic acid - see section 6.24), and thus should
be stored in a desiccator. Others (for example, sodium hydroxide)
readily absorb atmospheric carbon dioxide either when solid or in
solution, while phosphate buffer solutions are notorious for
permitting the growth of bacteria (often visible as a cloudy
precipitate).
Where chemicals or primary standards, such as drugs, are obtained
from secondary sources, it is important to have some idea of the
purity of the sample. Useful information can often be obtained by
carrying out a simple thin-layer chromatographic analysis, and the
ultraviolet spectrum can also be valuable. It is also possible to
measure the absorbance of a solution of the drug and compare the
result with tabulated specific absorbance values (the absorbance of a
1% (w/v) solution in a cell of 1-cm path length, see section 4.5.1).
For example, the specific absorbances for the drug colchicine in
ethanol are 730 and 350 at 243 nm and 425 nm, respectively. Thus, a
10 mg/l solution in ethanol should give absorbance readings of
0.73 and 0.35 at 243 nm and 425 nm, respectively, in a cell of 1-cm
path length. However, this procedure does not rule out the presence of
impurities with similar relative molecular masses and specific
absorbance values.
4.1.3 Balances and pipettes
Balances for weighing reagents or standards and automatic and
semi-automatic pipettes must be kept clean and checked for accuracy
regularly. Semi-automatic pipettes are normally calibrated to measure
aqueous fluids (relative density about 1), and should not be used
for organic solvents or other solutions with relative densities or
viscosities greatly different from those of water. Positive
displacement pipettes should be used for very viscous fluids, such as
whole blood. Accuracy can easily be tested by weighing or dispensing
purified (distilled or deionized) water; the volumes of 1.0000 g of
distilled water at different temperatures are given in Table 9. Low
relative humidities may give rise to static electrical effects,
particularly with plastic weighing boats, which can influence the
weight recorded.
Table 9. Volume of 1.0000 g of distilled water at different
temperatures
Temperature Volume Temperature Volume
(°C) (ml) (°C) (ml)
15 1.0020 24 1.0037
16 1.0021 25 1.0039
17 1.0023 26 1.0042
18 1.0025 27 1.0045
19 1 0026 28 1.0047
20 1.0028 29 1.0050
21 1.0030 30 1.0053
22 1.0032 31 1.0056
23 1.0034 32 1.0059
When preparing important reagents or primary standards,
particular attention should be paid to the relative molecular masses
(molecular weights) of salts and their degree of hydration (water of
crystallization). A simple example is the preparation of a cyanide
solution with a cyanide ion concentration of 50 mg/l. Potassium
cyanide has a relative molecular mass of 65.1 while that of the
cyanide ion is 26.0. A solution with a cyanide ion concentration of
50 mg/l is therefore equivalent to a potassium cyanide concentration
of 50 × 65.1/26.0 mg/l, i.e., 125.2 mg/l. Particular care should be
taken when weighing out primary calibration standards, and the final
weight plus tare (weighing boat) weight should be recorded.
4.1.4 Chemically pure water
Tapwater or well water is likely to contain dissolved material
which renders it unsuitable for laboratory use, so it is essential
that any water used for the preparation of reagents or standard
solutions is purified by distillation or deionization using a
commercial ion-exchange process. The simplest procedure is
distillation using an all-glass apparatus (glass distilled). The
distillation should not be allowed to proceed too vigorously otherwise
impurities may simply boil over into the distillate. Potassium
permanganate and sodium hydroxide (each at about 100 mg/l) added to
the water to be distilled will oxidize or ionize volatile organic
compounds or nitrogenous bases, and thus minimize contamination of the
purified water. If highly purified water is required then water
already distilled can be redistilled (double distilled). The pH of
distilled water is usually about 4 because of the presence of
dissolved carbon dioxide.
4.1.5 Quality assurance
Known positive and negative specimens should normally be analysed
at the same time as the test sample. A negative control (blank) helps
to ensure that false positives (owing to, for example, contaminated
reagents or glassware) are not obtained. Equally, inclusion of a true
positive serves to check that the reagents have been prepared properly
and have maintained their stability. Suspected false positive tests
should be repeated using glassware freshly cleaned with an organic
solvent such as methanol and/or purified water. In general, all
glassware, particularly test-tubes, should be rinsed in tapwater
immediately after use. This should be followed by rigorous cleaning in
warm laboratory detergent solution, then rinsing in tapwater and in
purified water before air-drying. Badly contaminated glassware can be
soaked initially in concentrated sulfuric acid (relative density 1.83)
containing 100 g/litre potassium dichromate (acid/dichromate, chromic
acid). However, this mixture is extremely dangerous, and treatment
with a modern laboratory detergent is usually all that is needed.
Quantitative tests require even more vigilance to ensure accuracy
and precision (reproducibility). When a new batch of a standard
solution is prepared it is prudent to compare the results obtained in
analysing a material of known concentration with those given by an
earlier batch or an external source to ensure that errors have not
been made in preparation. As in other areas of clinical laboratory
practice, it is important to organize an internal quality control
scheme for all quantitative procedures, and to participate in external
quality assurance schemes whenever possible.
4.1.6 Recording and reporting results
All results should be recorded on laboratory worksheets together
with the date, the name of the analyst, the name of the patient, and
other relevant information, including the number and nature of the
specimens received for analysis, and the tests performed. (An example
of a laboratory worksheet is given in Fig. 3). It is advisable to
allocate to each specimen a unique identifying number as it is
received in the laboratory, and to use this number when referring to
the tests performed using this specimen. Ultraviolet spectra,
calibration graphs and other documents generated during an analysis
should always be kept for a period of time after the results have been
reported. The recording of results of colour tests and thin-layer
chromatographic analyses is more difficult, and is discussed in
subsequent sections. Doubtful or unusual results should always be
discussed with senior staff. When reporting the results of tests in
which no compounds were detected in plasma/serum or in urine, the
limit of sensitivity of the test (detection limit) should always be
known, at least to the laboratory, and the scope of generic tests (for
example, for benzodiazepines or opioids) should be defined.
In analytical toxicology, SI mass units should be used to report
the results of quantitative analyses. The femtogram (fg)= 10-15 g,
picogram (pg) = 10-12 g, nanogram (ng)= 10-9 g, microgram (µg)=
10-6 g, milligram (mg) = 10-3 g, gram (g) and kilogram (kg)= 103 g
are the preferred units of mass, and the litre (l) is the preferred
unit of volume. Other units of concentration, mg %, mg/dl, µg/ml and
ppm (parts per million), are often encountered in the literature. It
is useful to remember that: 1 mg/l = 1 ppm = 1 µg/ml = 0.1 mg % =
0.1 mg/dl.
Some clinical chemistry departments report analytical toxicology
results in SI molar units (µmol/l, mmol/l, etc.). A list of conversion
factors is given in Annex 2. This is an area with great potential for
confusion, and care must be taken to ensure that the clinician is
fully aware of the units in which quantitative results are reported.
4.2 Colour tests
Many drugs and other poisons, if present in sufficient
concentration and in the absence of interfering compounds, give
characteristic colours with appropriate reagents. Some of these tests
are, for practical purposes, specific, but compounds containing
similar functional groups will also react, and thus interference from
other poisons, metabolites or contaminants is to be expected. Further
complications are that colour description is very subjective, even in
people with normal colour vision, while the colours produced usually
vary in intensity or hue with concentration, and may also be unstable.
Many of these tests can be performed satisfactorily in clear
glass test-tubes. However, use of a spotting tile (a white glazed
porcelain tile with a number of shallow depressions or wells in its
surface) gives a uniform background against which to assess any
colours produced, and also minimizes the volumes of reagents and
sample that need to be used. Colour tests feature prominently in the
monographs (section 6), where common problems and sources of
interference in particular tests are emphasized. When performing
colour tests it is always important to analyse concurrently with the
test sample:
(a) a reagent blank, i.e., an appropriate sample known not to contain
the compound(s) of interest; if the test is to be performed on
urine, then blank (analyte-free) urine should be used, otherwise
water is adequate;
(b) a known positive sample at an appropriate concentration. If the
test is to be performed on urine, then ideally urine from a
patient or volunteer known to have taken the compound in question
should be used. However, this is not always practicable and then
spiked urine (blank urine to which a known amount of the compound
under analysis has been added) should be used.
4.3 Pretreatment of samples
4.3.1 Introduction
Although many of the tests described in this manual can be
performed directly on biological fluids or other aqueous solution,
some form of sample pretreatment is often required. With plasma
and serum, a simple form of pretreatment is protein precipitation
by vortex-mixing with, for example, an aqueous solution of
trichloroacetic acid, followed by centrifugation to produce a clear
supernatant for analysis. Hydrolysis of some compounds, including
possibly conjugated metabolites in urine (sulfates and glucuronides),
either by heating with acid or by treatment with an enzyme
preparation, is also employed. This either gives a reactive compound
for the test (as with benzodiazepines and paracetamol) or enhances
sensitivity (as with laxatives and morphine).
4.3.2 Solvent extraction
Liquid-liquid extraction of drugs and other lipophilic poisons
from the specimen into an appropriate, water-immiscible, organic
solvent, usually at a controlled pH, is widely used in analytical
toxicology. Solvent extraction removes water and dissolved interfering
compounds, and reduction in volume (by evaporation) of the extract
before analysis provides a simple means of concentrating the compounds
of interest and thus enhancing sensitivity.
Some form of mechanical mixing of the aqueous and organic phases
is normally necessary. Of the methods available, vortex-mixing is the
quickest and the most efficient for relatively small volumes. Rotary
mixers capable of accepting tubes of up to 30 ml in volume are
valuable for performing relatively large volume extracts of plasma/
serum, urine, or stomach contents, and serve to minimize the risk of
emulsion formation. Centrifugation in a bench-top centrifuge, again
capable of accepting test-tubes of up to 30 ml in volume and attaining
speeds of 2000-3000 rev/min, is normally effective in separating the
phases so that the organic extract can be removed. Ideally, the
centrifuge should have a sealed motor unit (which is flashproof) and
tubes should be sealed to minimize both the risk of explosion from
ignition of solvent vapour and the risks associated with
centrifugation of infective specimens. Finally, filtration of the
organic extract through silicone-treated phase-separating paper
prevents contamination of the extract with small amounts of aqueous
phase.
Commercial prebuffered extraction tubes (so-called solid-phase
extraction) are now widely used for liquid-liquid extraction,
especially in preparing urine extracts for drug screening (see section
5.2.3). Such tubes have the advantage that a wide range of basic
compounds, including morphine, and weak acids, such as barbiturates,
can be extracted in a single step. However, they are relatively
expensive and cannot be reused.
4.3.3 Microdiffusion
Microdiffusion is also a form of sample purification and relies
on the liberation of a volatile compound (hydrogen cyanide in the case
of cyanide salts) from the test solution held in one compartment of an
enclosed system such as the specially constructed Conway apparatus
(Fig. 1). The volatile compound is subsequently trapped using an
appropriate reagent (sodium hydroxide solution in the case of hydrogen
cyanide) held in a separate compartment.
The cells are normally allowed to stand for 2-5 hours at room
temperature for the diffusion process to be completed. The analyte
concentration is subsequently measured in a portion of the trapping
solution either by spectrophotometry or by visual comparison with
standards analysed concurrently in separate cells. The Conway
apparatus is normally made from glass, but polycarbonate must be used
with fluorides since hydrogen fluoride etches glass. The cover is
often smeared with petroleum jelly or silicone grease to ensure an
airtight seal. In order to carry out a quantitative assay at least
eight cells are needed: one blank, three calibration samples, two test
samples and two positive controls. It is important to clean the
diffusion apparatus carefully after use, possibly using an
acid/dichromate mixture (see section 4.1.5), rinsing it in distilled
water before drying.
4.4 Thin-layer chromatography
Thin-layer chromatography (TLC) involves the movement by
capillary action of a liquid phase (usually an organic solvent)
through a thin, uniform layer of stationary phase (usually silica gel,
SiO2) held on a rigid or semi-rigid support, normally a glass,
aluminium or plastic sheet or plate. Compounds are separated by
partition between the mobile and stationary phases. TLC is relatively
inexpensive and simple to perform, and can be a powerful qualitative
technique when used together with some form of sample pretreatment,
such as solvent extraction. However, some separations can be difficult
to reproduce. The interpretation of results can also be very
difficult, especially if a number of drugs or metabolites are present.
TLC of solvent extracts of urine, stomach contents or scene
residues forms the basis of the drug screening procedure outlined in
section 5.2.3, and is also recommended for the detection and
identification of a number of compounds described in the monographs
(section 6). TLC can also be used as a semiquantitative technique, as
described in the monograph on coumarin anticoagulants (section 6.35).
The aim of this section is to provide practical information on
the use of TLC in analytical toxicology. More general information on
the theory and practice of TLC can be found in the references listed
in the Bibliography.
4.4.1 Preparation of TLC plates
The stationary phase is normally a uniform film (0.25 mm in
thickness) of silica gel (average particle size 20 µm). Plates usually
measure 20 × 20 cm, although smaller sizes can also be used. Some
commercially available plates incorporate a fluorescent indicator, and
this may be useful in locating spots prior to spraying with
visualization reagents. Prior soaking of the plate in methanolic
potassium hydroxide and drying may improve the chromatography of some
basic compounds using certain solvent systems but, generally, addition
of concentrated ammonium hydroxide (relative density 0.88) to the
mobile phase has the same effect (section 5.2.3). High-performance TLC
(HPTLC) plates have a smaller average particle size (5-10 µm) and
greater efficiency than conventional plates. Reversed-phase plates,
which have a hydrophobic moiety (usually C2, C8 or C18) bonded to
the silica matrix, are also available. However, HPTLC and reversed-
phase plates are more expensive and have a lower sample capacity than
conventional plates, and are not recommended for the procedures
outlined in this manual.
TLC plates can be prepared in the laboratory from silica gel
containing an appropriate binding agent and glass plates measuring
20 × 20 × 0.5 cm. It is important to ensure that the plates are clean
and free from grease. The silica gel is first mixed with twice its own
weight of water to form a slurry. The slurry is then quickly applied
to the glass plate using a commercial spreader to form a film 0.25 mm
in thickness. Small amounts of additives such as fluorescent markers
can be included if required. The plates are dried in air and should be
kept free of moisture prior to use. The quality of such home-made TLC
plates should be carefully monitored; activation (i.e., heating at
100°C for 30 minutes prior to use) may be helpful in maintaining
performance. Dipping techniques, whereby glass plates are coated by
dipping into a slurry of silica and then dried, give very variable
results and are not recommended. In general, home-made plates tend to
give silica layers that are much more fragile than those of
commercially available plates and chromatographic performance tends to
be much less reproducible. Experience suggests that it is best to use
one particular brand of commercially available plates. However, even
with commercial plates dramatic batch-to-batch variations in retention
and sensitivity to certain spray reagents may still be encountered.
4.4.2 Sample application
Some commercial plates are supplied with special adsorbent layers
to simplify application of the sample. Normally, however, the sample
is placed directly on to the silica-gel layer. The plate should be
prepared by marking the origin with a light pencil line at least 1 cm
from the bottom of the plate - care should be taken not to disturb the
silica surface in any way. A line should then be scored on the plate
10 cm above the origin to indicate the optimum position of the solvent
front; other distances may be used if required. It is advisable when
using 20 × 20-cm plates to score columns 2 cm in width vertically up
the plate with, say, a pencil since this minimizes edge effects, as
discussed in section 4.4.3.
The samples and any standards should be applied carefully at the
origin in the appropriate columns, using a micropipette or syringe so
as to form spots no more than than 5 mm in diameter. If larger spots
are produced, resolution will be impaired when the chromatogram is
developed. The volume of solvent applied should be kept to a minimum;
typically 5-10 µl of solution containing about 10 µg of analyte.
Sample extracts reconstituted as appropriate should be applied first,
followed by the standards or mixtures of standards; this sequence
minimizes the risk of cross-contamination. Glass capillaries intended
for use in melting-point apparatus can easily be drawn out in the
flame of a microburner to give disposable micropipettes with a very
fine point. Ideally, the solvent used in applying the sample should be
the same as that used to develop the chromatogram, but this is not
always practicable; methanol will usually prove satisfactory. The
plate may be heated with a hair-drier, for example, to increase the
speed of evaporation of the spotting solvent, but it must be allowed
to cool before development starts and there is a risk of loss of
volatile analytes such as amfetamines.
4.4.3 Developing the chromatogram
Glass TLC development tanks are available from many suppliers and
normally have a ground-glass rim which forms an airtight seal with a
glass cover plate. A small amount of silicone lubricant jelly may be
used to secure the seal. Some tanks have a well at the bottom which
reduces the amount of solvent required. Most of the procedures in this
manual recommend the use of plates and tanks of standard size, but
smaller tanks are advantageous if smaller plates are used. All tanks
should be lined with filter-paper or blotting paper on three sides
and the solvent should be added at least 30 minutes before the
chromatogram is to be developed. This helps to produce an atmosphere
saturated with solvent vapour, which in turn aids reproducible
chromatography. Some TLC mobile phases consist of a single solvent but
most are mixtures; possibly the most widely used mobile phase in
analytical toxicology is ethyl acetate/methanol/concentrated ammonium
hydroxide (EMA; see section 5.2.3). It is important to prepare mobile
phases daily, since their composition may change with time because of
evaporation or chemical reaction. In particular, loss of ammonia, not
only from the mobile phase but also from opened reagent bottles,
causes many problems.
The chromatogram is developed by placing the loaded plate in the
uniformly saturated tank, ensuring that the level of the solvent is
above the bottom edge of the silica layer on the plate but below the
level of the spots applied to the plate, and quickly replacing the
lid. The chromatogram should be observed to ensure that the solvent
front is rising up the plate uniformly. Usually the solvent front will
show curvature at the edges of the plate; more serious curvature or
bowing may be observed if the tank atmosphere is not sufficiently
saturated with solvent vapour. This effect can be minimized by
dividing the plate into 2-cm columns as indicated in section 4.4.2.
The chromatogram should be allowed to develop for the intended
distance, usually 10 cm from the origin. The plate should then be
taken from the tank, placed in a fume cupboard or under a fume hood
and allowed to dry. This process may be enhanced by blowing warm air
(from a hair-drier) over the plate for several minutes until all
traces of solvent have been removed. This can be especially important
with ammoniacal mobile phases, since the presence of residual ammonia
affects the reactions observed with certain spray reagents.
4.4.4 Visualizing the chromatogram
When the chromatogram has been developed and the plate dried, the
chromatogram should be examined under ultraviolet light (at 254 nm and
366 nm) and the positions of any fluorescent compounds (spots) noted.
This stage is essential if a fluorescent marker has been added to the
silica, as any compounds present appear as dark areas against a
fluorescent background. However, in analytical toxicology the use of
chromagenic chemical detection reagents generally gives more useful
information, as discussed in section 5.2.3, and in the appropriate
monographs (section 6). Plates can be dipped in reagent but, unless
special precautions are taken, the structure of the silica tends to be
lost and the chromatogram destroyed. Thus, the reagent is normally
lightly applied as an aerosol, using a commercial spray bottle
attached to a compressed air or nitrogen line. Varying the line
pressure varies the density of the aerosol and thus the amount of
reagent reaching the chromatogram in a given time.
Normally, the plate should be sprayed in an inverted position,
since this avoids the risk of excess reagent being drawn up the plate
by capillary action and destroying the lower part of the chromatogram.
Glass plates can be used to mask portions of the plate if columns are
to be sprayed with different reagents. Alternatively, if plastic or
aluminium plates are used then columns can be cut up and sprayed
separately. The appearance of certain compounds may change with time,
and it is important to record results as quickly and carefully as
possible, noting any changes with time. A standardized recording
system is valuable for reference purposes, as discussed in section
5.2.3. Many spray reagents are extremely toxic - always use a fume
cupboard or hood when spraying TLC plates.
4.4.5 Retention factors
TLC results are usually recorded as retention factors. The
retention factor (Rf) is defined as follows:
Distance travelled from the origin by the analyte
Rf =
Distance travelled from the origin by the solvent front
A more convenient value is Rf × 100 (hRf), especially if a standard
length of chromatogram of 10 cm is always used, since then hRf is
equal to the distance in millimetres travelled from the origin by the
analyte.
There are many factors that influence the reproducibility of hRf
values including (1) the TLC plate itself, (2) the amount of analyte
applied to the plate, (3) the development distance, (4) the degree of
tank saturation, and (5) the ambient temperature. However, the
influence of these factors can be minimized if standard (reference)
compounds are analysed together with each sample. For unknown
substances, it is a relatively simple procedure to obtain a corrected
hRf value from a calibration graph constructed from experimentally
observed values of sample and reference compounds. However, a further
complicating factor is that the chromatography of compounds that
originate from biological extracts may be different from that of the
pure substances because of interferences from additional material
present in sample extracts (matrix effects) (see section 5.2.3).
4.5 Ultraviolet and visible spectrophotometry
A number of the quantitative methods described in the
monographs (section 6) employ ultraviolet (UV) (200-400 nm) or visible
(400-800 nm) spectrophotometry. The major problem encountered with
this technique is interference, and some form of sample purification,
such as solvent extraction or microdiffusion (see section 4.3), is
usually employed. The spectrophotometer may be of the single-beam or
double-beam type. With a single-beam instrument, light passes from the
source through a monochromator and then via a sample cell to the
detector. With double-beam instruments, light from the monochromator
passes through a beam-splitting device and then via separate sample
and reference cells to the detector. Double-beam instruments with
automated wavelength scanning and a variety of other features are also
available.
4.5.1 The Beer-Lambert law
In spectrophotometry, the relationship between the intensity of
light entering and leaving a cell is governed by the Beer-Lambert law,
which states that, for a solution with an absorbing solute in a
transparent solvent, the fraction of the incident light absorbed is
proportional to the number of solute molecules in the light path,
i.e.,
log10Io/I = kcb
where
Io is the incident light intensity,
I is the transmitted light intensity,
c is the solute concentration (g/l),
b is the path length (cm),
k is the absorptivity of the system.
The constant k is a fundamental property of the solute, but also
depends on temperature, wavelength and solvent. The term log10Io/I
is known as absorbance (A) and, for dilute solutions only, is linearly
related to both solute concentration and path length. In older
textbooks it was known as optical density (OD) or extinction
coefficient (E), but these terms are now obsolete. The specific
absorbance (A1%, 1 cm) is the absorbance of a 1% (w/v) (10 g/litre)
solution of the solute in a cell of 1-cm path length, and is usually
written in the shortened form A11.
4.5.2. Spectrophotometric assays
With all types of spectrophotometer it is important to ensure
that the monochromator is correctly aligned. This can be checked by
observing the absorbance maxima (lambdamax) of a known reference
solution or material. For example, a holmium oxide glass filter has
major peaks at a number of important wavelengths (241.5 nm, 279.4 nm,
287.5 nm, 333.7 nm, 360.9 nm, 418.4 nm, 453.2 nm, 536.2 nm and
637.5 nm). A simple method of checking the photometric accuracy is to
measure the absorbance of an acidic potassium dichromate solution (see
Table 10).
It is important that the cells used in the spectrophotometer are
of the correct specification and that they are scrupulously clean.
Glass and certain types of plastic cells are suitable for measurements
in the visible region (> 400 nm), but only fused silica or quartz
cells should be used for UV work (< 400 nm). Normally, cells of 1-cm
path length are used, but cells of 2-cm or 4-cm path length can
sometimes enhance sensitivity.
Table 10. Photometric calibration using potassium dichromate
(60.00 mg/l) in aqueous sulfuric acid (0.005 mol/l)a
Wavelength Specific absorbance
(nm) (A11)
235 124.5
257 144.0
313 48.6
350 106.6
a Values from British Pharmacopoeia, London, Her Majesty's
Stationery Office, 1980.
Double-beam spectrophotometers have the advantage that
background absorbance from reagents, solvents, etc., can be allowed
for by including a blank (analyte-free) extract in the reference
position. Normally, an extract of blank plasma or serum is used in the
reference cell, but purified water can be used in certain assays. In
high sensitivity work, it is important to use matching cells, i.e.,
cells with similar absorbance values, for the test and reference
measurements. Pairs of matched cells can be purchased and should be
kept together.
As mentioned previously, a major worry in many
spectrophotometric assays is the risk of interference from co-ingested
drugs or other compounds. However, some information as to the purity
of a sample extract can often be obtained by examining the UV
absorption spectrum. While this can be done most easily using an
instrument with a built-in scanning facility, it can also be performed
manually on simpler instruments. UV spectra of extracts of stomach
contents or scene residues can also give useful qualitative
information, and can be used as an adjunct to the drug screening
procedure described in section 5.2. However, such an approach is only
practical with an instrument with a built-in scanning facility.a
a UV absorption spectra of many compounds of interest are given
in Clarke's isolation and identification of drugs (Moffat,
1986) (see Bibliography, section 1), but again care is needed to
ensure that the pH/solvent combination employed is the same as
that used to produce the reference spectrum.
5 Qualitative tests for poisons
Many difficulties may be encountered when performing qualitative
tests for poisons, especially if laboratory facilities are limited.
The poisons may include gases, such as carbon monoxide, drugs,
solvents, pesticides, metal salts, corrosive liquids (acids, alkalis)
and natural toxins. Some poisons may be pure chemicals and others
complex natural products. Not surprisingly, there is no comprehensive
range of tests for all poisons in all samples.
When certain compounds are suggested by the history or clinical
findings, simple tests may be performed using the procedures given in
the monographs (section 6). However, in the absence of clinical or
other evidence to indicate the poison(s) involved, a defined series of
tests (a screen) is needed. It is usually advisable to perform this
series of tests routinely, since circumstantial evidence of poisoning
is often misleading. Similarly, the analysis should not end after the
first positive result, since additional unsuspected compounds may be
present.
The sequence of analyses outlined in section 5.2 will detect and
identify a number of poisons in commonly available specimens (urine,
stomach contents, and scene residues, i.e., material such as tablets
or suspect solutions found with or near to the patient) using a
minimum of apparatus and reagents. The compounds detected include many
that give rise to nonspecific features, such as drowsiness, coma or
convulsions, and which will not be indicated by clinical examination
alone. Poisons for which specific therapy is available, such as
acetylsalicylic acid and paracetamol, are also included. The analysis
takes about 2 hours and may be modified to incorporate common local
poisons if appropriate tests are available.
5.1 Collection, storage and use of specimens
5.1.1 Clinical liaison
Good liaison between the clinician and the analyst is of vital
importance if the results of a toxicological analysis are to be useful
(see section 2). Ideally, this liaison should commence before the
specimens are collected, and any special sample requirements for
particular analytes noted. At the very least, a request form should be
completed to accompany the specimens to the laboratory. An example of
such a form is given in Fig. 2.
Before starting an analysis it is important to obtain as much
information about the patient as possible (medical, social and
occupational history, treatment given, and the results of laboratory
or other investigations), as discussed in sections 2 and 3. It is also
important to be aware of the time that elapsed between ingestion or
exposure and the collection of samples, since this may influence the
interpretation of results. All relevant information about a patient
gathered in conversation with the clinician, nurse, or poisons
information service should be recorded in the laboratory using the
external request form (Fig. 2) or a suitably modified version of this
form.
5.1.2 Specimen transport and storage
Specimens sent for analysis must be clearly labelled with the
patient's full name, the date and time of collection, and the nature
of the specimen if this is not self-evident. This is especially
important if large numbers of patients have been involved in a
particular incident, or a number of specimens have been obtained from
one patient. Confusion frequently arises when one or more blood
samples are separated in a local laboratory and the original
containers are discarded. When the plasma/serum samples are forwarded
subsequently to the toxicology laboratory for analysis, it can be
difficult, if not impossible, to ascertain which is which.
The date and time of receipt of all specimens by the laboratory
should be recorded and a unique identifying number assigned to each
specimen (see section 4.1.6). Containers of volatile materials, such
as organic solvents, should be packaged separately from biological
specimens to avoid the possibility of cross-contamination. All
biological specimens should be stored at 4°C prior to analysis, if
possible, and ideally any specimen remaining after the analysis should
be kept at 4°C for 3-4 weeks in case further analyses are required. In
view of the medicolegal implications of some poison cases (for
example, if it is not clear how the poison was administered or if the
patient dies) then any specimen remaining should be kept (preferably
at -20°C) until investigation of the incident has been concluded.
5.1.3 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 if chromatographic techniques are used. A 50-ml
specimen from an adult, collected in a sealed, sterile container, is
sufficient for most purposes; no preservative should be added. The
sample should be obtained as soon as possible, ideally before any drug
therapy is initiated. However, drugs such as tricyclic antidepressants
(amitriptyline, imipramine) cause urinary retention, and thus a very
early specimen may contain insignificant amounts of poison.
Conversely, little poison may remain in specimens taken many hours or
days later, even though the patient may be very ill, as in acute
paracetamol poisoning. If the specimen is obtained by catheterization
there is a possibility of contamination with lidocaine. If syrup of
ipecacuanha has been given in an unsuccessful attempt to induce emesis
there is a possibility of emetine being present in the urine.
5.1.4 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
required to carry out a wide range of tests; no preservative should be
added. This can be a very variable sample and additional procedures
such as homogenization followed by filtration and/or centrifugation
may be required to produce a fluid amenable to analysis. However, 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. Note
that emetine from syrup of ipecacuanha may be present, especially in
children (section 2.2.1).
5.1.5 Scene residues
It is important that all bottles or other containers and other
suspect materials found with or near the patient (scene residues) are
retained for analysis if necessary since they may be related to the
poisoning episode. There is always the possibility that the original
contents of containers have been discarded and replaced either with
innocuous material or with more noxious ingredients such as acid,
bleach or pesticides. Note that it is always best to analyse
biological specimens in the first instance if possible.
A few milligrams of scene residues are usually sufficient for the
tests described here. Dissolve solid material in a few millilitres of
water or other appropriate solvent. Use as small an amount as possible
in each test, in order to conserve sufficient for possible further
tests.
5.1.6 Blood
Blood (plasma or serum) is normally reserved for quantitative
assays but for some poisons, such as carbon monoxide and cyanide,
whole blood has to be used for qualitative tests. For adults, a 10-ml
sample should be collected in a heparinized tube on admission. In
addition, a 2-ml sample should be collected in a fluoride/oxalate
tube, if ethanol poisoning is suspected. Note that tubes of this type
available commercially contain the equivalent of about 1 g/l fluoride,
whereas about 10 g/l fluoride (40 mg sodium fluoride per 2 ml of
blood) is needed to inhibit fully microbial action in such
specimens. The use of disinfectant swabs containing alcohols (ethanol,
propan-2-ol) should be avoided. The sample should be dispensed with
care: the vigorous discharge of blood though a syringe needle can
cause sufficient haemolysis to invalidate a serum iron or potassium
assay.
In general, there are no significant differences in the
concentrations of poisons between plasma and serum. However, if a
compound is not present to any extent within erythrocytes, the use of
lysed whole blood will result in considerable dilution of the
specimen. On the other hand, some poisons, such as carbon monoxide,
cyanide and lead, are found primarily in erythrocytes and thus whole
blood is needed for such measurements. A heparinized whole blood
sample will give either whole blood or plasma as appropriate. The
space above the blood in the tube (headspace) should be minimized if
carbon monoxide poisoning is suspected.
5.2 Analysis of urine, stomach contents and scene residues
If any tests are to influence immediate clinical management, the
results must be available within 2-3 hours of receipt of the specimen.
Of course, a positive result does not in itself confirm poisoning,
since such a result may arise from incidental or occupational exposure
to the poison in question or the use of drugs in treatment. In some
cases, the presence of more than one poison may complicate the
analysis, and examination of further specimens from the patient may be
required. A quantitative analysis carried out on whole blood or plasma
may be needed to confirm poisoning, but this may not be possible if
laboratory facilities are limited. It is important to discuss the
scope and limitations of the tests performed with the clinician
concerned, and to maintain high standards of laboratory practice (see
section 4.1), especially when performing tests on an emergency basis.
It may be better to offer no result rather than misleading data based
on an unreliable test. In any event, it is valuable to have a
worksheet to record the analytical results. An example of such a sheet
is given in Fig. 3.
The qualitative scheme given below, possibly modified to suit
local needs, should be followed in every case unless there are good
reasons (such as insufficient sample) for omitting part of the screen,
since this will provide a good chance of detecting any poisons
present. The scheme has three parts: physical examination, colour
tests and thin-layer chromatography, and is designed primarily for the
analysis of urine samples. However, most of the tests and some
additional ones are also applicable, with due precautions, to stomach
contents and scene residues. Some compounds and groups of compounds
not normally detected using this procedure are listed in Table 11.
Simple tests for many of these compounds are given in the monographs
(section 6).
Table 11. Some compounds not detected in urine by the drug
screening procedure
Group Compound
Inorganic ions arsenic, barium, bismuth, borate, bromide,
cadmium, copper, cyanide, fluoride, lead, lithium,
mercury, sulfide, thallium
Organic chemicals camphor, carbon disulfide, carbon monoxide, carbon
tetrachloride, dichloromethane, ethylene glycol,
formates, oxalates, petroleum distillates,
phenols, tetrachloroethylene, toluene, 1,1,1-
trichloroethane
Drugs benzodiazepines, coumarin anticoagulants,
dapsone, digoxin, ethchlorvynol, glyceryl
trinitrate, meprobamate, monoamine oxidase
inhibitors, theophylline, tolbutamide
Pesticides carbamate pesticides, chloralose, chlorophenoxy
herbicides, dinitrophenol pesticides,
fluoroacetates, hydroxybenzonitrile herbicides,
methyl bromide, organochlorine pesticides,
organophosphorus pesticides, pentachlorophenol
5.2.1 Physical examination of the specimen
Urine
High concentrations of some drugs or metabolites can impart
characteristic colours to urine (Table 12). Deferoxamine or methylene
blue given in treatment may colour urine red or blue, respectively.
Strong-smelling poisons such as camphor, ethchlorvynol and methyl
salicylate can sometimes be recognized in urine since they are
excreted in part unchanged. Acetone may arise from metabolism of
propan-2-ol. Turbidity may be due to underlying pathology (blood,
microorganisms, casts, epithelial cells), or to 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.
Chronic therapy with sulfonamides may give rise to yellow or greenish
brown crystals in neutral or alkaline urine. Phenytoin, primidone, and
sultiame form crystals in urine following overdosage, while
characteristic colourless crystals of calcium oxalate form at neutral
pH after ingestion of ethylene glycol (Fig. 4).
Table 12. Some possible causes of coloured urine
Colour Possible cause
Brown or black nitrobenzene, phenols, rhubarb (liver failure)
(intensifying on standing)
Yellow or orange cascara, fluorescein, phenolphthalein,
nitrofurantoin, senna
Wine red or brown aloin, phenothiazines, phenytoin, phenolphthalein,
quinine, warfarin (haematuria)
Blue or green amitriptyline, indometacin, phenols
Stomach contents and scene residues
Some characteristic smells associated with particular substances
are listed in Table 13. Many other compounds (for example,
ethchlorvynol, methyl salicylate, paraldehyde, phenelzine) also have
distinctive smells. Very low or very high pH may indicate ingestion of
acid or alkali, while a green/blue colour suggests the presence of
iron or copper salts. Microscopic examination using a polarizing
microscope may reveal the presence of tablet or capsule debris. Starch
granules used as a filler in Some tablets and capsules are best
identified using crossed polarizing filters, when they appear as
bright grains marked with a dark Maltese cross.
Table 13. Characteristic smells associated with particular poisonsa
Smell Possible cause
Bitter almonds cyanide
Fruity alcohols (including ethanol), esters
Garlic arsenic, phosphorus
Mothballs camphor
Pears