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    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
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    (c) World Health Organization 1995

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    Health Organization in preference to others of a similar nature that
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    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).

         Systme internationale (International System; SI) mass units
    (mg/l, g/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, mol/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, -blockers, digoxin, opioids
    Hypertension             Anticholinergics, sympathomimetics
    Hypotension              Ethanol, hypnotics/sedatives, opioids,
                               tranquillizers, many other compounds
    Arrhythmias              -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 g/l (serum)              )   Deferoxamine

    Lead                     1 mg/l (whole blood,                 DMSAc/DMPSd/
                               adults)                            Sodium calcium edetate
    Cadmium                  20 g/l (whole blood)                DMSA
    Mercury                  100 g/l (whole blood)               DMSA/DMPS
    Arsenic                  200 g/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 (g)=
    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, g/ml and
    ppm (parts per million), are often encountered in the literature. It
    is useful to remember that: 1 mg/l = 1 ppm = 1 g/ml = 0.1 mg % =
    0.1 mg/dl.

         Some clinical chemistry departments report analytical toxicology
    results in SI molar units (mol/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).

    FIGURE 1

         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 m). 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 m) 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
    100C 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 l of solution containing about 10 g 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.

    FIGURE 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 4C prior to analysis, if
    possible, and ideally any specimen remaining after the analysis should
    be kept at 4C 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 -20C) 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

    FIGURE 3

    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
                                                                                     
    
    FIGURE 4

    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               chloral
    Petrol              petroleum distillates (may be vehicle in
                          pesticide formulation)
    Phenolic            disinfectants, phenols
    Stale tobacco       nicotine
    Shoe polish         nitrobenzene
    Sweet               chloroform and other halogenated hydrocarbons
                                                                        

    a    Take care: specimens containing cyanide may give off hydrogen
         cyanide, especially if acidified - not everyone can detect
         hydrogen cyanide by smell. Similarly sulfides evolve hydrogen
         sulfide - the ability to detect hydrogen sulfide (rotten egg
         smell) is lost at higher concentrations.

         Undegraded tablets or capsules and any plant remains or specimens
    of plants thought to have been ingested should be examined separately.
    The local poisons information service will normally have access to
    publications or other aids to the identification of tablets or
    capsules by weight, markings, colour, shape and possibly other
    physical features.

    5.2.2  Colour tests

         The nine qualitative tests described here are based on simple
    colour reactions and cover a number of important drugs and other
    poisons. Full descriptions of these tests are given in the respective
    monographs (section 6), together with details of common sources of
    interference and detection limits. Other tests, such as the Reinsch
    test for antimony, arsenic, bismuth and mercury, are not discussed
    further here, but full details are given in the respective monographs.

         Of the tests outlined (Table 14), that for salicylates such as
    acetylsalicylic acid (aspirin) (Trinder's test, also known as the
    ferric chloride test) is best performed on urine rather than stomach
    contents or scene residues, since acetylsalicylic acid itself does not
    react unless hydrolysed. Most of the others may be performed using
    either specimen, but the tests for chlorates and other oxidizing
    agents and for ferrous or ferric iron can only be carried out on
    stomach contents or scene residues. Examples of the colours obtained
    in these tests are given in Plates 1-8.

    5.2.3  Thin-layer chromatography

         The aim of the scheme outlined below is to obtain as much
    information as possible in a short time and with a minimum of sample.
    Drugs and, in some cases, metabolites present are extracted from the
    sample into an organic solvent under acidic and alkaline conditions.
    The extracts are analysed by thin-layer chromatography (TLC) on one
    plate using a single solvent system. The basic extract is acidified
    during the evaporation stage to minimize loss of volatile bases such
    as amfetamines. If the sample volume is limited, the acidic and basic
    extractions can be performed sequentially on the same portion of the
    specimen. However, it is important that the pH change between
    extractions is accomplished satisfactorily. Extracts of stomach
    contents may contain fatty material, which makes chromatographic
    analysis difficult, and purification by re-extraction into aqueous
    acid or alkali may be required.

         Although simple examination of the developed chromatogram under
    ultraviolet light (254 nm and 366 nm) may reveal the presence of
    fluorescent compounds such as quinine, the use of a number of spray
    (visualization) reagents widens the scope of the analysis and
    increases the confidence of any identifications.

    Table 14.  Recommended qualitative colour tests
                                                                        

    1.  Salicylic acid (including acetylsalicylic acid 
         (aspirin)) - Trinder's test

        Add 100 l of Trinder's reagent (a mixture of 40 g of mercuric
        chloride in 850 ml of water and 120 ml of aqueous hydrochloric
        acid (1 mol/l) and 40 g of hydrated ferric nitrate, diluted to
        1 l with water) to 2 ml of urine and mix for 5 seconds. A
        violet colour indicates the presence of salicylates.

        If only stomach contents or scene residues are available,
        hydrolyse by heating with 0.5 mol/l hydrochloric acid on a
        boiling water-bath for 2 minutes, and neutralize with 0.5 mol/l
        sodium hydroxide before performing the test (see section 6.99).

        If a positive result is obtained in this test, carry out a
        quantitative assay on plasma/serum (see section 6.99).

    2.  Phenothiazines - FPN test

        Add 1 ml of FPN reagent (a mixture of 5 ml of aqueous ferric
        chloride solution (50 g/litre), 45 ml of aqueous perchloric
        acid (200 g/kg) and 50 ml of aqueous nitric acid
        (500 ml/litre)) to 1 ml of sample and mix for 5 seconds.
        Colours ranging from pink to red, orange, violet or blue
        suggest the presence of phenothiazines.

        Positive results should be confirmed by thin-layer
        chromatography (section 5.2.3).

    3.  Imipramine and related compounds - Forrest test

        Add 1 ml of Forrest reagent (a mixture of 25 ml of aqueous
        potassium dichromate (2 g/l), 25 ml of aqueous sulfuric acid
        (300 ml/l), 25 ml of aqueous perchloric acid (200 g/kg) and
        25 ml of aqueous nitric acid (500 ml/l)) to 0.5 ml of sample
        and mix for 5 seconds. A yellow-green colour deepening through
        dark green to blue indicates the presence of imipramine or
        related compounds.

        Positive results should be confirmed by thin-layer
        chromatography (section 5.2.3).

    4.  Trichloro compounds (including chloral hydrate, chloroform,
        dichloralphenazone and trichloroethylene) - Fujiwara test

        To three 10-ml tubes, add respectively 1-ml portions of (a)
        sample, (b) purified water (blank test -  essential), and (c)
        aqueous trichloroacetic acid (10 mg/l). Add 1 ml of sodium
        hydroxide solution (5 mol/l) and 1 ml of pyridine to each tube,
        mix carefully and heat in a boiling water-bath for 2 minutes.
        An intense red/purple colour in the top (pyridine) layer of
        tube (a) (as in tube (c)) indicates the presence of trichloro
        compounds; tube (b) should show no coloration.

    5.  Paracetamol, phenacetin - o-cresol/ammonia test

        Add 0.5 ml of concentrated hydrochloric acid to 0.5 ml of
        sample, heat in a boiling water-bath for 10 minutes and cool.
        Add 1 ml of aqueous o-cresol solution (10 g/l) to 0.2 ml of the
        hydrolysate, add 2 ml of ammonium hydroxide solution (4 mol/l),
        and mix for 5 seconds. A strong blue to blue-black colour,
        which forms immediately, indicates the presence of paracetamol
        or phenacetin.

        If a positive result is obtained in this test carry out a
        quantitative assay for paracetamol on plasma or serum (see
        section 6.83).

    6.  Paraquat, diquat - dithionite test

        Add 0.5 ml of aqueous ammonium hydroxide (2 mol/l) to 1 ml of
        test solution, mix for 5 seconds and add about 20 mg of solid
        sodium dithionite. A strong, blue to blue-black colour
        indicates paraquat; diquat gives a yellow-green colour, but
        this is insignificant in the presence of paraquat.

        If the colour fades on continued agitation in air and is
        restored by adding further sodium dithionite, paraquat or
        diquat is confirmed.

    7.  Ethanol and other volatile reducing agents - dichromate test

        Apply 50 l of potassium dichromate (25 g/l in aqueous sulfuric
        acid (500 ml/l)) to a strip of glass-fibre filter-paper and
        insert the paper in the neck of a test-tube containing 1 ml of
        urine. Lightly stopper the tube and place in a boiling water-
        bath for 2 minutes. A change in colour from orange to green
        indicates the presence of volatile reducing substances.

        If a positive result is obtained in this test carry out a
        quantitative assay for ethanol on blood (see section 6.46).

    8.  Chlorates and other oxidizing agents - diphenylamine testa

        Carefully add 0.5 ml of diphenylamine (10 g/l in concentrated
        sulfuric acid) to 0.5 ml of filtered stomach contents or scene
        residue. A strong blue colour which develops rapidly indicates
        the presence of oxidizing agents.

    9.  Ferrous and ferric iron - ferricyanide/ferrocyanide testa

        To 50 l of filtered stomach contents or scene residue add
        100 l of aqueous hydrochloric acid (2 mol/l) and 50 l of
        aqueous potassium ferricyanide solution (10 g/l), To a further
        50 l of sample add 100 l of hydrochloric acid and 50 l of
        potassium ferrocyanide solution (10 g/l). A deep blue
        precipitate with potassium ferricyanide or ferrocyanide
        indicates the presence of ferrous or ferric iron.

        If a positive result is obtained in this test carry out a
        quantitative assay for iron on serum (see section 6.61).
                                                                        

    a    Tests for use on stomach contents or scene residues only.

         The recommended TLC visualization reagents are as follows:

    1.   Mercurous nitrate reagent (acidic extract), which gives white
         spots with a grey centre on a darker background with barbiturates
         and related compounds such as glutethimide.

    2.   Acidified iodoplatinate reagent (basic extract), which gives
         mainly purple, blue or brown spots with a range of basic and
         neutral drugs and metabolites. Note that some authors recommend
         neutral iodoplatinate, which is more stable and which gives
         similar reactions with many basic drugs; this is oversprayed with
         sulfuric acid (500 ml/l) which facilitates reactions with neutral
         compounds such as caffeine and phenazone (from
         dichloralphenazone).

    3.   Mandelin's reagent (basic extract) gives colours ranging from
         blue and green to orange and red with a variety of basic
         compounds. Some, especially tricyclic antidepressants such as
         amitriptyline and nortriptyline, give fluorescent spots if viewed
         under ultraviolet light (366 nm) after spraying with this
         reagent.

    4.   Sulfuric acid (500 ml/l) (basic extract) alone gives red, purple
         or blue spots with many phenothiazines and their metabolites.
         This is especially valuable since some phenothiazines
         (chlorpromazine, for example) are given therapeutically in
         relatively high doses and have many metabolites, which can give a
         very confusing picture if unrecognized.

         Of course, many additional mobile phase and spray reagent
    combinations could be used as well as, or in place of, those suggested
    here, and details of some are given in the references listed in the
    Bibliography and in the monographs (section 6). For example,
    methanol:concentrated ammonium hydroxide (99:1.5) (methanol:ammonia,
    MA) is widely used in the analysis of basic drugs, and is especially
    useful in the detection of morphine and related opioids (see section
    6.73). Of the spray reagents, Marquis' reagent gives a variety of
    colours with different basic drugs, and is again especially valuable
    for the detection of morphine and other opioids which give blue/violet
    colours.

         In all cases, the colour obtained from a particular compound may
    vary depending on concentration, the presence of co-eluting compounds,
    the duration and intensity of spraying, and the type of silica used in
    the manufacture of the plate, among other factors. Some compounds may
    show a gradation or even a change in colour from the edge of the spot
    towards the centre (usually a concentration effect), while the
    intensity or even the nature of the colour obtained may vary with
    time. A further problem is that the interpretation and recording of
    colour reactions are very subjective. It is thus important to analyse
    authentic compounds, ideally on the same plate as the sample extracts.
    Even so, compounds present in sample extracts sometimes show slightly
    different chromatograms from the pure compounds owing to the presence
    of co-extracted material. Interfering neutral compounds, especially
    fatty acids from stomach contents, can be removed by back-extraction
    of acidic or basic compounds into dilute base or acid, respectively
    (the neutral compounds stay in the organic extract), followed by
    re-extraction into organic solvent.

         Detailed records of each analysis should be kept, not only for
    medicolegal purposes, but also to establish a reference data bank to
    aid in the interpretation of results. This can be used to supplement
    the data given in Table 15 and elsewhere, and has the advantage of
    being generated in the laboratory actually involved in analysing the
    specimens.

         The recommended TLC screening system is as follows.

    Qualitative analysis

         Applicable to urine, stomach contents or scene residues.

        Table 15.  Thin-layer chromatography hRf values (eluent EMA) and colour reactions (rim colour) with spray reagents
                                                                                                                            

    Compound                      hRf                                   Visualization reagent
                                                                                                                            

                                            Mercurous        Acidified            Mandelin's       Mandelin's      Sulfuric
                                            nitrate          iodoplatinate        reagent          reagent           acid
                                                             (visible light)a     (UV 366 nm)a     (500 ml/l)
                                                                                                                            

    3-acetylmorphine              24        --               blue                 --               --                --
    6-acetylmorphine              48        --               blue                 --               --                --
      (diamorphine metabolites)
    N-acetylprocainamide          --        --               blue                 --               --                --
      (procainamide metabolite)
    amfetamine                    44        --               blue                 --               --                --
    aminophenazone                61        --               blue                 --               --                --
    amiodarone                    82        --               blue                 --               --                --
    amitriptyline                 70        --               blue                 pale blue        blue (yellow)     --
    amobarbital                   40        white-grey       --                   --               --                --
    atropine                      25        --               blue                 --               --                --
    barbital                      33        white-grey       --                   --               --                --
    benzoctamine                  77        --               purple               grey             --                --
    benzoylecgonine               2         --               blue                 --               --                --
      (cocaine metabolite)
    brallobarbital                29        white-grey       --                   --               --                --
    brucine                       26        --               blue                 --               --                --
    butobarbital                  39        white-grey       --                   --               --                --
    caffeine                      50        --               blue                 --               --                --
    carbamazepine                 57        --               blue                 pale blue        green             --
    chloroquine                   52        --               blue                 --               pale blue         --
    chlorpromazine                70        --               purple               red              --                red
    chlorprothixene               74        --               purple               pink             orange            pink
                                                                                                   (yellow)
                                                                                                                            

    Table 15.  (Con't)
                                                                                                                            

    Compound                      hRf                                   Visualization reagent
                                                                                                                            

                                            Mercurous        Acidified            Mandelin's       Mandelin's      Sulfuric
                                            nitrate          iodoplatinate        reagent          reagent           acid
                                                             (visible light)a     (UV 366 nm)a     (500 ml/l)
                                                                                                                            

    clomethiazole                 74        --               blue                 --               --                --
    clomipramine                  72        --               purple               green-blue       pale blue         --
    cocaine                       77        --               purple               --               --                --
    codeine                       35        --               black                pale blue        --                --
    cotinine                      40        --               blue                 --               --                --
      (nicotine metabolite)
    cyclizine                     68        --               blue                 yellow           --                --
    cyclobarbital                 35        white-grey       --                   --               --                --
    desipramine                   41        --               purple               dark blue        --                --
    dextropropoxyphene            80        --               purple               grey             blue (brown)      --
    diamorphine (heroin)b         51        --               blue                 --               --                --
    dibenzepin                    57        --               purple               pale blue        yellow            --
    dihydrocodeine                27        --               black                pale blue        --                --
    diphenhydramine               68        --               blue                 yellow           yellow            --
    dosulepin                     65        --               purple               pale blue        pale blue         --
    doxepin                       65        --               purple               brown            orange            --
    emetine                       62        --               purple               white            --                --
    ephedrine                     27        --               blue                 --               --                --
    ethylmorphine                 36        --               blue                 --               --                --
    fenfluramine                  60        --               blue                 --               --                --
    glutethimide                  78        white-grey       --                   --               --                --
    haloperidol                   74        --               purple               --               --                --
    hexobarbital                  51        white-grey       --                   --               --                --
    imipramine                    67        --               purple               dark blue        pale blue         --
    iproniazid                    42        --               blue                 violet           --                --
    isoniazid                     29        --               blue                 --               --                --
                                                                                                                            

    Table 15.  (Con't)
                                                                                                                            

    Compound                      hRf                                   Visualization reagent
                                                                                                                            

                                            Mercurous        Acidified            Mandelin's       Mandelin's      Sulfuric
                                            nitrate          iodoplatinate        reagent          reagent           acid
                                                             (visible light)a     (UV 366 nm)a     (500 ml/l)
                                                                                                                            

    levorphanol                   41        --               brown                white            --                --
    lidocaine                     80        --               blue                 --               --                --
    maprotiline                   35        --               blue                 blue             --                --
    mefenamic acid                14        --               --                   blue-grey        --                --
    metamfetamine                 35        --               blue                 --               --                --
    methadone                     77        --               brown                white            --                --
    methaqualone                  78        --               --                   --               green             --
    methylephedrine               35        --               blue                 --               --                --
    methyprylon                   63        white-grey       --                   --               --                --
    metixene                      70        --               purple               blue             purple            --
    morphine                      20        --               blue                 --               --                --
    nicotine                      61        --               blue                 --               --                --
    norephedrine                  28        --               blue                 --               --                --
      (ephedrine metabolite)
    norpethidine                  34        --               purple               --               --                --
      (pethidine metabolite)
    nortriptyline                 45        --               blue                 pale blue        blue (yellow)     --
      (amitriptyline metabolite)
    opipramol                     38        --               blue                 yellow           pale green        --
    orphenadrine                  70        --               blue                 yellow           pale blue         --
    oxycodone                     60        --               purple               white            purple            --
    pentazocine                   72        --               brown                grey             pale blue         --
    perphenazine                  43        --               --                   red              --                red
    pethidine                     62        --               purple               --               --                --
    phenazone (from               44        --               blue                 --               --                --
      dichloralphenazone)
                                                                                                                            

    Table 15.  (Con't)
                                                                                                                            

    Compound                      hRf                                   Visualization reagent
                                                                                                                            

                                            Mercurous        Acidified            Mandelin's       Mandelin's      Sulfuric
                                            nitrate          iodoplatinate        reagent          reagent           acid
                                                             (visible light)a     (UV 366 nm)a     (500 ml/l)
                                                                                                                            

    phenelzine                    83        --               --                   pink             --                --
    phenobarbital                 29        white-grey       --                   --               --                --
    phenytoin                     39        white-grey       --                   --                                 --
    primidone                     40        white-grey       --                   --               --                --
    procainamide                  39        --               blue                 --               --                --
    prochlorperazine              54        --               blue                 red              --                red
    promazine                     65        --               --                   red-brown        --                red
    promethazine                  65        --               purple               red              purple            red
    propranolol                   52        --               blue                 blue             --                --
    protriptyline                 41        --               blue                 orange           brown             --
                                                                                  (purple)         (green)           --
    quinidine                     52        --               purple               --               blue
    quinine                       52        --               purple               --               blue
    secobarbital                  42        white-grey       --                   --               --
    strychnine                    33        --               blue                 --               --                --
    theophylline                  10        white-grey       --                   --               --                --
    thiopental                    49        white-grey       --                   --               --                __
    thioridazine                  67        --               purple-brown         blue-green       --                purple
    tofenacin                     --        --               blue                 yellow           pale blue         --
      (orphenadrine metabolite)
                                                                                                                            

    Table 15.  (Con't)
                                                                                                                            

    Compound                      hRf                                   Visualization reagent
                                                                                                                            

                                            Mercurous        Acidified            Mandelin's       Mandelin's      Sulfuric
                                            nitrate          iodoplatinate        reagent          reagent           acid
                                                             (visible light)a     (UV 366 nm)a     (500 ml/l)
                                                                                                                            

    tolbutamide                   12        --               blue                 --               --                --
    tranylcypromine               60        --               --                   violet           --                --
    trazodone                     68        --               purple               purple           blue              --
    trimipramine                  80        --               blue                 dark blue        --                --
    trifluoperazine               56        --               purple               pink             --                pink
    verapamil                     74        --               blue                 --               --                --
                                                                                                                            

    a   With certain compounds, there may be a colour difference between the centre and the outer part of the spot;
        this is indicated by the mention of a second colour in parentheses,
    b   Diamorphine itself is not found in urine, but is detected as monoacetyl morphine and morphine conjugates
        Reagents and equipment

    1.   Aqueous hydrochloric acid (1 mol/l).

    2.   Aqueous sodium hydroxide (0.5 mol/l).

    3.   Ammonium chloride buffer. Saturated aqueous ammonium chloride
         adjusted to pH 9 with concentrated ammonium hydroxide (relative
         density 0.88).

    4.   Hydrochloric acid (2 ml/l in methanol).

    5.   Ethyl acetate:methanol:concentrated ammonium hydroxide (EMA)
         (85:10:5) (relative density 0.88).

    6.   Mercurous nitrate reagent. Place 1 g of mercurous nitrate in
         100 ml of purified water and add concentrated nitric acid
         (relative density 1.42) until the solution is clear.

    7.   Acidified iodoplatinate reagent. Mix 0.25 g of platinic chloride,
         5 g of potassium iodide and 5 ml of concentrated hydrochloric
         acid (relative density 1.18) in 100 ml of purified water.

    8.   Mandelin's reagent. Suspend 1 g of finely powdered ammonium
         vanadate in 100 ml of concentrated sulfuric acid (relative
         density 1.86).  Shake well before use.

    9.   Aqueous sulfuric acid (500 ml/l).

    10.  Silica gel thin-layer chromatography plate (20  20 cm, 20 m
         average particle size; see section 4.4.1).

    Standards

    All 1 g/l in chloroform:

    1.   Acidic drugs mixture (amobarbital, mefenamic acid, phenobarbital,
         theophylline).

    2.   Basic drugs mixture (amitriptyline, codeine, nicotine,
         nortriptyline).

    3.   Phenothiazine mixture (perphenazine, trifluoperazine,
         thioridazine).

    Methods

    1.   Acidic extract (extract A)

         (a)  To 10 ml of urine in a 30-ml glass centrifuge tube add 1 ml
              of dilute hydrochloric acid and 10 ml of chloroform.

         (b)  Shake on a mechanical shaker for 5 minutes, centrifuge in a
              bench centrifuge for 10 minutes and transfer the lower,
              organic layer to a 15-ml tapered glass tube.

         (c)  Evaporate the extract to dryness on a water-bath at 60C
              under a stream of compressed air.

    2.   Basic extract (extract B)

         (a)  To a further 10 ml of urine in a 30-ml glass tube, add 2 ml
              of ammonium chloride buffer and 10 ml of chloroform:propan-
              2-ol (9:1).

         (b)  Shake on a mechanical shaker for 5 minutes, centrifuge in a
              bench centrifuge for 10 minutes and transfer the lower,
              organic layer to a 15-ml tapered glass tube.

         (c)  Add 0.5 ml of methanolic hydrochloric acid (to minimize
              losses of volatile bases; see section 6.1) and evaporate the
              extract to dryness in a water-bath at 60C under a stream of
              compressed air.

    3.   Purification of extracts of stomach contents

         (a)  Prior to the solvent evaporation stage, add 5 ml of aqueous
              sodium hydroxide solution to extract A, and 5 ml of aqueous
              hydrochloric acid to extract B.

         (b)  Shake on a mechanical shaker for 5 minutes, centrifuge in a
              bench centrifuge for 10 minutes and discard both organic
              layers.

         (c)  Add 5 ml of aqueous hydrochloric acid to the aqueous residue
              from extract A, and 5 ml of ammonium chloride buffer to the
              aqueous residue from extract B, and re-extract into
              chloroform or chloroform:propan-2-ol as in methods 1 and 2
              above.

    Thin-layer chromatography

    1.   Divide the plate into eight columns by scoring with a pencil (see
         section 4.4.2). Lightly draw a pencil line about 1 cm from the
         bottom of the plate to indicate the origin, and score a
         horizontal line 10 cm from the origin to indicate the limit of
         development, as shown in Fig. 5.

    2.   Reconstitute each extract in 100 l of chloroform:propan-2-ol and
         apply 25 of extract A and 3 portions of 25 l of extract B at the
         origin of the plate, as shown in Fig. 5.

    3.   Apply 10 l of the respective standard mixtures to the plate, as
         shown in Fig. 5.

    4.   Ensure that the plate is dry, and then develop the chromatogram
         using ethyl acetate:methanol:concentrated ammonium hydroxide
         (EMA) (saturated tank, see section 4.4.3).

    5.   Remove the plate and dry under a stream of air in a fume cupboard
         or under a fume hood.

    6.   View the plate under ultraviolet light (254 nm and 366 nm) and
         note any fluorescent spots.

    7.   Invert the plate and spray each portion with the visualization
         reagents as shown in Fig. 6. Take care to mask with a clean glass
         plate those portions of the plate not being sprayed.

    Take care - all the spray reagents used are very toxic.
    Spraying must be performed in a fume cupboard or under
    an efficient fume hood.

    8.   View the plate again under ultraviolet light (254 nm and 366 nm)
         and note any fluorescent spots, especially in the portion sprayed
         with Mandelin's reagent.

    9.   If necessary, the colours obtained with Mandelin's reagent can be
         enhanced by heating the plate in an oven at 100C for 10 minutes.

    Results

         hRf values and reactions with the spray reagents of some of the
    compounds of interest are given in Table 15 and illustrated in Plates
    9-12.

    FIGURE 5

    .FIGURE 5a;SUP2_06.BMP
    .FIGURE 5b;SUP2_07.BMP

    .FIGURE 5c;SUP2_08.BMP
    .FIGURE 5d;SUP2_09.BMP

    .FIGURE 5e;SUP2_10.BMP

    .FIGURE 5f;SUP2_11.BMP

    .FIGURE 5g;SUP2_12.BMP

    .FIGURE 5h;SUP2_13.BMP

    .FIGURE 5i;SUP2_14.BMP

    Plate 9. An example of a chromatogram obtained from the analysis of an
    acidic and a basic urine extract from a patient following the
    ingestion of codeine and methadone. The plate is divided into four
    sections for the application of separate visualization reagents: A -
    mercurous nitrate; B - acidified iodoplatinate; C - Mandelin's
    reagent; D - sulfuric acid. The acidic sample extract (TA) has been
    applied at the origin in section A and run with test mixture S1
    containing: amobarbital (1), phenobarbital (2), and theophylline (3).
    The "basic" extract (TB) has been applied at the origin in sections B,
    C and D and run with test mixtures: S2 containing amitriptyline (4),
    nicotine (5), nortriptyline (6), codeine (7), and mefenamic acid (8);
    and S3 containing thioridazine (9), trifluoperazine (10), and
    perphenazine (11).

    Note that a complex pattern of drug and metabolites is obtained for
    the basic sample extract (TB) visualized with iodoplatinate (B) and
    Mandelin's reagent (C). No response is seen using sulfuric acid (D).
    No response is seen for the acidic sample extract (TA) visualized
    using mercurous nitrate in section A.

    .FIGURE 5j;SUP2_15.BMP

    Plate 10. An example of a chromatogram obtained from the analysis of
    an acidic and a basic urine extract from a patient following the
    ingestion of phenobarbital and methadone. The plate is divided into
    four sections for the application of separate visualization reagents:
    A - mercurous nitrate; B acidified iodoplatinate; C - Mandelin's
    reagent; D - sulfuric acid. The acidic sample extract (TA) has been
    applied at the origin in section A and run with test mixture S1
    containing: amobarbital (1), phenobarbital (2), and theophylline (3).
    The basic extract (TB) has been applied at the origin in sections B, C
    and D and run with test mixtures: S2 containing amitriptyline (4),
    nicotine (5), nortriptyline (6), codeine (7), and mefenamic acid (8);
    and S3 containing thioridazine (9), trifluoperazine (10), and
    perphenazine (11).

    Note that the phenobarbital is clearly visualized in the acidic urine
    extract (TA) using the mercurous nitrate spray (A), whereas methadone
    and its metabolites are best visualized in the basic urine extract
    (TB) with iodoplatinate (B). No distinct response is seen with the
    remaining two sprays, Mandelin's reagent (C) and sulfuric acid (D).

    .FIGURE 5k;SUP2_16.BMP

    Plate 11. An example of a chromatogram obtained from the analysis of
    an acidic and a basic urine extract from a patient following the
    ingestion of a phenothiazine (thioridazine) overdose. The plate is
    divided into four sections for the application of separate
    visualization reagents: A -mercurous nitrate; B - acidified
    iodoplatinate; C - Mandelin's reagent; D - sulfuric acid. The acidic
    sample extract (TA) has been applied at the origin in section A and
    run with test mixture S1 containing: amobarbital (1), phenobarbital
    (2), and theophylline (3). The basic extract (TB) has been applied at
    the origin in sections B, C and D and run with test mixtures: S2
    containing amitriptyline (4), nicotine (5), nortriptyline (6), codeine
    (7), and mefenamic acid (8); and S3 containing thioridazine (9),
    trifluoperazine (10), and perphenazine (11).

    Note that a complex pattern of drug and metabolites is obtained for
    the basic sample extract (TB) visualized with iodoplatinate (B),
    Mandelin's reagent (C) and sulfuric acid (D). No response is seen with
    mercurous nitrate (A) for the acidic sample extract (TA). The pattern
    of many spots and distinct colours with sulfuric acid is typical of
    phenothiazine-type compounds, but the pattern is quite dissimilar to
    that seen for the pure compound (thioridazine).

    .FIGURE 5l;SUP2_17.BMP

    Plate 12. An example of a chromatogram obtained from the analysis of
    an acidic and a basic urine extract from a patient following the
    ingestion of the tricyclic antidepressant, dosulepin. The plate is
    divided into four sections for the application of separate
    visualization reagents: A -mercurous nitrate; B - acidified
    iodoplatinate; C - Mandelin's reagent; D - sulfuric acid. The acidic
    sample extract (TA) has been applied at the origin in section A and
    run with test mixture S1 containing: amobarbital (1), phenobarbital
    (2), and theophylline (3). The basic extract (TB) has been applied at
    the origin in sections B, C and D and run with test mixtures: S2
    containing amitriptyline (4), nicotine (5), nortriptyline (6), codeine
    (7), and mefenamic acid (8); and S3 containing thioridazine (9),
    trifluoperazine (10), and perphenazine (11).

    Note that dosulepin and its metabolites are visualized in the basic
    urine extract (TB) most clearly using the iodoplatinate spray (B). No
    response is seen with the other sprays (C and D), or with mercurous
    nitrate spray (A).

    FIGURE 6

         Benzodiazepines and their metabolites may appear as light green
    or yellow spots under ultraviolet light (366 nm) before the mercurous
    nitrate column (acidic extract) is sprayed, but these compounds are
    not considered further here (see section 6.11). Quinine (often from
    bitter drinks), carbamazepine and their metabolites give fluorescent
    spots (254 nm and 366 nm) before the columns containing the basic
    extracts are sprayed, and also undergo characteristic reactions with
    some of the spray reagents. These compounds are relatively easy to
    identify, as are tricyclic antidepressants (amitriptyline, and
    imipramine). Others such as nicotine and its metabolites (normally
    from tobacco), caffeine (from caffeinated beverages) and lidocaine
    (from catheter lubricant) occur frequently and should be recognizable
    with practice.

         In difficult cases it may be useful to calculate the hRf value
    for unknown compounds (section 4.4.5) and to compare the findings with
    reference values (see Bibliography).

         Plates 9-12 give some examples of the spot shapes and colours
    that should be expected for the standards and for some other commonly
    occurring compounds. Even if the interpretation of the chromatography
    plate is relatively straightforward, it is important to record
    systematically the data generated. This can be done by photographing
    or photocopying the plate (taking great care to clean the photocopier
    and any other surfaces very carefully afterwards), but it is as easy
    to record spot positions and shapes on a standard form such as that
    illustrated in Fig. 7.

         Note the position of the yellow-brown streak near the top of the
    plate observed with Mandelin's reagent (visible light) on analysis of
    blank urine. Colours, including those observed under ultraviolet
    light, and any temporal changes, can be noted either in writing or by
    using coloured pencils.

         To ensure reproducible chromatography, attention should be given
    to the factors discussed in section 4.4.3, especially the use of
    saturated tanks and the need to ensure that the concentrated ammonium
    hydroxide is of adequate strength (relative density 0.88, 330 g/l),
    both in the tank and in the reagent bottle. It is good practice to buy
    either small (500-ml) bottles or to transfer the contents of large
    (2.5-litre) bottles of ammonium hydroxide to 500-ml bottles ( with
     care), which can be kept tightly stoppered until needed.  Never use
    a batch of ethyl acetate:methanol:concentrated ammonium hydroxide
    mobile phase more than five times at room temperatures of 20-25C
    (fewer times at higher ambient temperatures).

    FIGURE 7

         Even when all due precautions are taken, it is invariably found
    that the chromatographic characteristics of sample extracts are
    different from those of pure compounds. In extreme cases, broad
    streaks may be obtained rather than discrete spots. This is often
    attributable to the presence of polyethylene glycol used as a vehicle
    in, for example, temazepam capsules and can be minimized by back-
    extraction of acidic or basic compounds into dilute base or acid,
    respectively, as described above.

    Sensitivity

         It is not possible to give detection limits for all the compounds
    under study. The experience of the analyst, the extraction efficiency,
    the spot density, the intensity of the chromogenic reaction with the
    spray reagents and even the type of silica gel used can all affect
    sensitivity. Nevertheless, a general limit of sensitivity of 1 mg/l is
    reasonable.

    5.2.4  Reporting the results

         The results of emergency analyses must be communicated direct to
    the clinician without delay, and should be followed by a written
    report as soon as possible. An example of an analytical toxicology
    report form is given in Fig. 8. Ideally, confirmation using a second,
    independent method, or failing this an independent duplicate, should
    be obtained before positive findings are reported. However, this may
    not always be practicable, especially if only simple methods are
    available. In such cases it is vital that the appropriate positive and
    negative controls have been analysed together with the specimen (see
    section 4.1.5).

         When reporting quantitative results it is important to state
    clearly the units of measurement used (SI mass units are preferable;
    see section 4.1.6). In addition, any information necessary to ensure
    that the clinical implications of the result are fully understood
    should be noted on the written report. The clinical features
    associated with poisoning by a number of compounds are given in the
    appropriate monographs (section 6). Information on additional
    compounds will usually be found in one of the clinical toxicology
    textbooks listed in the Bibliography.

         Although it is often easy for the analyst to interpret the
    results of analyses in which no compounds are detected, such results
    are sometimes difficult to convey to clinicians, especially in
    writing. It is important to give information as to the poisons
    excluded by the tests performed with all the attendant complications
    of the scope, sensitivity and selectivity of the analyses, and other
    factors such as sampling variations. Because of the potential

    FIGURE 8

    medicolegal and other implications of any toxicological analysis, it
    is important not to use laboratory jargon such as "negative" or
    sweeping statements such as "absent" or "not present".

         The phrase "not detected" should convey precisely the laboratory
    result, especially when accompanied by a description of the specimen
    analysed and the limit of sensitivity of the test (detection limit).
    However, it can still be difficult to convey the scope of analyses,
    such as the thin-layer chromatography screen for acidic and basic
    drugs discussed above. Even with Trinder's test for example, - a
    relatively simple test normally used to detect acetylsalicylic acid
    ingestion (Table 14) - a number of other salicylates including, of
    course, salicylic acid itself also react. One way of giving at least
    some of this information in a written report is to list the compounds
    or groups of compounds normally detected by commonly used procedures.
    If these groups are listed on the back of the report then it is
    relatively simple to refer to the qualitative tests performed by
    number and thus to convey at least some of the information required.
    An example of a grouping system based on that used in the analytical
    toxicology laboratory worksheet (see Fig. 3) is given in Table 16.

    5.2.5  Summary

         It should be clear that performing a qualitative poisons screen
    involves more than simply analysing specimens as they arrive in the
    laboratory and reporting the bare facts of the analysis. The suggested
    scheme of operation is summarized in Table 17.

    Table 16.  Qualitative test groups (see also Fig. 3)
                                                                        

    Group            Compounds
                                                                        

    1      Salicylates (including acetylsalicylic acid (aspirin),
             4-aminosalicylic acid, methyl salicylate and salicylic acid)
    2      Phenothiazines (including chlorpromazine, perphenazine,
             prochlorperazine, promazine, promethazine and thioridazine)
    3      Imipramine and related compounds (including clomipramine,
             desipramine and trimipramine)
    4      Trichloro compounds (including chloral hydrate, chloroform,
             dichloralphenazone and trichloroethylene
    5      Paracetamol
    6      Paraquat and diquat
    7      Volatile reducing agents (including ethanol and methanol)
    8      Strong oxidizing agents (including bromates, chlorates,
             hypochlorites, nitrates and nitrites)
    9      Iron
    10a    TLC acidic drugs (including barbiturates, glutethimide,
             methyprylon, phenytoin and primidone)
    10b    TLC basic drugs (including antihistamines (cyclizine and
             diphenhydramine), antimalarials (chloroquine and quinine),
             amfetamine, atropine, caffeine, carbamazepine,
             cardioactive drugs (lidocaine, propranolol, quinidine, and
             verapamil), clomethiazole, cocaine, ephedrine, haloperidol,
             methaqualone, opioids (codeine, dextropropoxyphene,
             diamorphine, dihydrocodeine, methadone, morphine and
             pethidine), orphenadrine, phenothiazines (chlorpromazine,
             perphenazine, prochlorperazine, promazine, promethazine and
             thioridazine), strychnine and tricyclic antidepressants
             (amitriptyline, clomipramine, doxepin, desipramine,
             dosulepin, imipramine, nortriptyline, protriptyline and
             trimipramine))
                                                                        

    Table 17.  Summary of the suggested analytical scheme
                                                                        

    Step                Action
                                                                        

    1      Assess urgency and nature of request. Confirm and record
           clinical details (see Fig. 2). Advise on collection of
           specimens.
    2      Check details of the specimens received, particularly dates and
           times in relation to suspected date and time of ingestion.
    3      Carry out preliminary physical examination of samples and scene
           residues (section 5.2.1)
    4      Perform simple tests for specific compounds if indicated by
           history or clinical examination (section 6). Also, if indicated
           by the history, perform quantitative measurements using plasma,
           serum or whole blood. Record results using laboratory worksheet
           (see Fig. 3).
    5      Carry out direct qualitative tests on urine, stomach contents
           or scene residues (sections 5.2.2 and 5.2.3). Record the
           results using laboratory worksheet (see Fig. 3).
    6      Prepare acidic and basic extracts of the sample for TLC
           (section 5.2.3). Spot sample extracts and standards (sample
           extracts first) and develop TLC plates in ethyl acetate:
           methanol: concentrated ammonium hydroxide (85:10:5) (EMA).
    7      Dry the plate and visualize the chromatogram in the sequence:
           (a) ultraviolet (254 nm and 366 nm), (b) spray reagents, and
           (c) ultraviolet (254 nm and 366 nm).
    8      Collate the results and prepare a written record as soon as
           possible (see Fig. 7). Are the results in keeping with the
           clinical condition of the patient (feasibility control)?
    9      Perform quantitative measurements using plasma/serum or whole
           blood if indicated. Carry out any necessary additional tests on
           the original, or additional, samples.
    10     Telephone urgent results to clinical staff, taking care to
           ensure that they are written down and that the clinical
           implications are fully understood. Issue written report (see
           Fig. 8).
                                                                        
    
.
Basic Analytical Toxicology INTOX Home Page

    6  Monographs - analytical and toxicological data

         These monographs give practical information on detecting and
    identifying some common poisons or groups of poisons (see Table 1). In
    order to simplify presentation, no original references have been
    given, but further details on individual entries are available in the
    references listed in the Bibliography. Reference is made as
    appropriate to specific aspects of laboratory practice (section 4) and
    to the poisons screening procedure (section 5.2). As noted previously,
    this procedure should usually be followed when there is no clinical or
    circumstantial evidence as to the poison(s) involved in a particular
    case.

    6.1  Amfetamine

         alpha-Methylphenethylamine; C9H13N; relative molecular mass, 135

    CHEMICAL STRUCTURE 1

         Amfetamine and its  N-methyl analogue, metamfetamine, are
    central nervous system stimulants and are widely abused.

         There is no simple qualitative test for amfetamine, but this and
    other similar compounds can be detected and identified by thin-layer
    chromatography of a basic solvent extract of urine, stomach contents
    or scene residues. However, the extract  must be acidified by
    addition of 0.5 ml of methanolic hydrochloric acid (2 ml/l) to prevent
    loss of volatile bases at the evaporation stage, as noted in section
    5.2.3.

    Clinical interpretation

         Oral or intravenous amfetamine overdosage may cause hyperthermia,
    convulsions, coma, and respiratory and/or cardiac failure, but death
    from acute amfetamine poisoning is comparatively rare. Treatment is
    generally symptomatic and supportive. Quantitative measurements in
    blood are not normally required in management.

    6.2  Aminophenazone

         Amidopyrine, aminopyrine, 4-dimethylamino-1,5-dimethyl-2-phenyl-
    4-pyrazolin-3-one; C13H17N3O; relative molecular mass, 231.

    CHEMICAL STRUCTURE 2

         Aminophenazone is an analgesic and antipyretic which is now
    little used since agranulocytosis and renal tubular necrosis may occur
    after therapeutic dosage. Ingestion of about 10 g can cause severe
    acute poisoning in an adult.

    Qualitative test

         Applicable to urine, stomach contents and scene residues.

     Reagents

    1.   Aqueous sodium hydroxide (1 mol/l).

    2.   Aqueous silver nitrate solution (100 g/l).

    3.   Aqueous hydrochloric acid (5 mol/l).

    4.   Potassium nitrite (solid).

    Method

    1.   Add 1 ml of sodium hydroxide solution to 5 ml of sample and add
         10 ml of chloroform.

    2.   Extract on a mechanical shaker for 5 minutes, centrifuge and
         discard the upper aqueous phase.

    3.   Filter the chloroform extract through phase-separating filter-
         paper (see section 4.3.2) into a clean tube, evaporate to dryness
         under a stream of compressed air or nitrogen, and reconstitute
         the residue in 1 ml of purified water.

    4.   Add 0.5 ml of silver nitrate solution to 0.5 ml of the
         reconstituted extract.

    5.   Add 1 ml of hydrochloric acid and about 1 mg of solid potassium
         nitrite to the remaining portion of the extract.

     Results

         On addition of silver nitrate (step 4), a blue solution which
    turns black on standing indicates aminophenazone. In step 5, potassium
    nitrite imparts a blue-violet colour which quickly fades.

         Aminophenazone can also be detected by thin-layer chromatography
    of a basic extract of urine, stomach contents or scene residues
    (section 5.2.3), and this should always be performed in addition to
    the test given above.

     Sensitivity

         Aminophenazone, 50 mg/l.

    Clinical interpretation

         Aminophenazone overdosage may cause hypotension, convulsions, and
    delirium. Treatment is symptomatic and supportive. Quantitative
    measurements in blood are not required in management.

    6.3  Amitriptyline

    3-(10,11-Dihydro-5 H-dibenzo [a,d]cyclohepten-5-ylidene)-
     N,N-dimethylpropylamine; C20H23N; relative molecular mass, 277

    CHEMICAL STRUCTURE 3

         Amitriptyline is a widely used tricyclic antidepressant. It is
    metabolized by  N-demethylation to nortriptyline, which is an
    antidepressant in its own right. Protriptyline is an analogue of
    amitriptyline.

         There is no simple qualitative test for amitriptyline, but this
    compound and other tricyclic antidepressants can be easily detected
    and identified by thin-layer chromatography of a basic solvent extract
    of urine, stomach contents or scene residues (see section 5.2.3).

    Clinical interpretation

         Acute poisoning with amitriptyline and other tricyclic
    antidepressants may cause dilated pupils, hypotension, hypothermia,
    cardiac arrhythmias, depressed respiration, coma, convulsions and
    cardiorespiratory arrest. Urinary retention is also a feature of
    poisoning with these compounds, and this may delay procurement of an
    appropriate specimen for analysis.

         Treatment is generally symptomatic and supportive. The use of
    antiarrhythmic agents should generally be avoided, but alkalinization
    using sodium bicarbonate may be employed. Quantitative measurements in
    blood are not normally required in management.

    6.4  Aniline

         Phenylamine; C6H5NH2; relative molecular mass, 93

         Aniline is used mainly as an intermediate in the manufacture of
    dyes and other chemicals. It is metabolized to  p-aminophenol and
     p-acetamidophenol, which are excreted in urine as sulfate and
    glucuronide conjugates. On hydrolysis of urine,  p-aminophenol is
    reformed, and can be detected using the  o-cresol/ammonia test.
    Aniline and other primary aromatic amines form diazo compounds with
    nitrous acid, which couple with 1-naphthylethylenediamine to form
    highly coloured derivatives. This reaction forms the basis of the
    confirmatory test described below.

    Qualitative test

         Applicable to urine.  o-Cresol/ammonia test.

    Reagents

    1.   Concentrated hydrochloric acid (relative density 1.18).

    2.   Aqueous  o-cresol (10 g/l).

    3.   Aqueous ammonium hydroxide (4 mol/l).

    Method

    1.   Add 0.5 ml of hydrochloric acid to 0.5 ml of sample, boil for 10
         minutes and cool.

    2.   Add 1 ml of  o-cresol solution to 0.2 ml of the hydrolysate.

    3.   Add 2 ml of ammonium hydroxide solution and mix for 5 seconds.

    Results

         A strong, royal blue colour developing rapidly indicates the
    presence of p-aminophenol. Metabolites of paracetamol (and thus
    phenacetin) and nitrobenzene also give  p-aminophenol on hydrolysis
    and so interfere. Ethylenediamine (from aminophylline, for example;
    see section 6.105) gives a green colour in this test.

    Sensitivity

          p-Aminophenol, 10 mg/l.

    Confirmatory test

         Applicable to stomach contents and scene residues.

    Reagents

    1.   Aqueous sodium nitrite solution (2 g/l, freshly prepared).

    2.   Aqueous hydrochloric acid (2 mol/l).

    3.   Aqueous ammonium sulfamate solution (10 g/l).

    4.   Aqueous  N-(1-naphthyl)ethylenediamine dihydrochloride solution
         (2 g/l, freshly prepared).

    Method

    1.   Mix 0.1 ml of sodium nitrite solution and 0.2 ml of dilute
         hydrochloric acid in a 5-ml test-tube.

    2.   Add 0.1 ml of sample, mix and allow to stand for 2 minutes.

    3.   Add 0.2 ml of ammonium sulfamate solution followed by 0.1 ml of
          N-(1-naphthyl)ethylenediamine dihydrochloride solution.

    Results

         A purple colour after 1 minute indicates the presence of aniline.

    Sensitivity

         Aniline, 10 mg/l.

    Clinical interpretation

         Poisoning with aniline usually results from inhalation or dermal
    absorption. Symptoms occur within 1-3 hours of exposure and include
    confusion, nausea, vomiting and diarrhoea, with convulsions, coma and
    hepatic and renal damage in severe cases. Haemolysis, red (wine)-
    coloured urine, and methaemoglobinaemia (dark chocolate-coloured
    blood) may also occur (section 3.2.2). Blood methaemoglobin can be
    measured but is unstable and the use of stored samples is unreliable.
    Hepatic and renal function tests are essential, however. Treatment may
    include intravenous methylene blue, but this is contraindicated in
    patients with glucose-6-phosphate dehydrogenase deficiency, since
    there is a high risk of inducing haemolysis.

    6.5  Antimony

         Trivalent and pentavalent salts of antimony (Sb) are used
    parenterally in the treatment of schistosomiasis and leishmaniasis.
    Antimony salts are also used in pigments and abrasives and for flame-
    proofing fabrics. As with arsenic, bismuth and mercury, antimony can
    be detected using the Reinsch test.

    Qualitative test

         Applicable to urine, stomach contents and scene residues.

    Reagents

    1.   Concentrated hydrochloric acid (relative density 1.18).

    2.   Aqueous hydrochloric acid (2 mol/l).

    3.   Copper foil or mesh (5  10 mm) or wire (2-3 cm).

    4.   Aqueous nitric acid (500 ml/l).

    Method

    1.   Immediately before use, clean the foil, mesh or wire in nitric
         acid until the copper acquires a bright surface.

    2.   Rinse the copper with purified water and add 10 ml of
         concentrated hydrochloric acid and 20 ml of test solution in a
         100-ml conical flask.

    3.   Heat on a boiling water-bath in a fume cupboard for 1 hour.
         Maintain the volume of the solution by adding dilute hydrochloric
         acid as necessary.

    4.   Cool and gently wash the copper with purified water.

    Results

         Staining on the copper can be interpreted as follows:

    purple black - antimony

    dull black - arsenic

    shiny black - bismuth

    silvery - mercury

    Selenium and tellurium may also give dark deposits, while high
    concentrations of sulfur may give a speckled appearance to the copper.

         An estimation of the concentration of antimony in the sample can
    be made by comparison of the deposit on the copper with that obtained
    from a solution containing a known concentration of the element.

    Sensitivity

         Antimony, about 2 mg/l.

    Confirmatory test

         Applicable to the stained (purple black) copper from the test
    above.

    Reagents

    1.   Aqueous potassium cyanide solution (100 g/l). Take care when
         using concentrated cyanide solutions.

    2.   Aqueous sodium sulfite solution (50 g/l, freshly prepared).

    3.   Aqueous nitric acid (3 mol/l).

    4.   Quinine/potassium iodide reagent. Dissolve 1 g of quinine sulfate
         in 100 ml of purified water containing 0.5 ml of concentrated
         nitric acid (relative density 1.42). When the quinine has
         completely dissolved, add 2 g of potassium iodide.

    Method

    1.   Place the copper in potassium cyanide solution and leave for 10
         minutes.

    2.   Wash any undissolved stain with purified water and add 1 ml of
         sodium sulfite solution and 1 ml of aqueous nitric acid.

    3.   Agitate frequently for 5 minutes and add 1 ml of purified water
         and 1 ml of quinine/potassium iodide reagent.

    Results

         Stains due to arsenic dissolve in potassium cyanide solution,
    while stains due to bismuth and antimony do not. Bismuth slowly forms
    an orange/brown suspension with quinine/potassium iodide.

    Sensitivity

         Antimony, about 2 mg/l.

    Clinical interpretation

         Parenteral administration of antimony salts may lead to
    cardiotoxicity; collapse and death from anaphylactic shock may also
    occur. Industrial poisoning is usually due to inhalation of antimony
    compounds either as fumes or dusts. The symptoms of acute oral
    antimony poisoning resemble those of acute arsenic poisoning and
    include abdominal pain, vomiting and diarrhoea. The measurement of
    blood antimony concentrations is useful in diagnosing acute poisoning,
    but this requires atomic absorption spectrophotometry.

    6.6  Arsenic

         A number of pesticides contain arsenic (As) in the form of
    arsenic acid, dimethylarsenic acid, and arsenite, arsenate and
    methanearsonate salts. Arsenical compounds are also used in
    pharmaceuticals and in the manufacture of ceramics and glass. Arsine
    (AsH3) gas is used in certain industrial processes and may also be
    liberated accidentally from other arsenical products.

         As with antimony, bismuth and mercury, arsenic can be detected
    and identified using the Reinsch test. The method described below for
    the quantitative assay to measure urinary arsenic concentrations is a
    modified Gutzeit procedure. In summary, arsine is generated by
    reaction of arsenic-containing compounds in the sample with nascent
    hydrogen. The arsine is carried in a stream of hydrogen through a lead
    acetate-impregnated filter (to remove sulfides), and arsenic is
    trapped in a bubbler by a solution of silver diethyldithiocarbamate in
    pyridine.

    Qualitative test

         Applicable to urine, stomach contents and scene residues.

         Reinsch test - see antimony monograph (section 6.5).

    Results

         Staining on the copper can be interpreted as follows:

    purple black - antimony

    dull black - arsenic

    shiny black - bismuth

    silvery - mercury

    Selenium and tellurium may also give dark deposits, while high
    concentrations of sulfur may give a speckled appearance to the copper.

         An estimation of the concentration of arsenic in the sample can
    be made by comparison of the deposit on the copper with that obtained
    from a solution containing a known concentration of the element.

    Sensitivity

         Arsenic, about 5 mg/l.

    Confirmatory test

         Applicable to the stained (dull black) copper from the test
    above.

    Reagent

         Aqueous potassium cyanide solution (100 g/l).  Take care when
     using concentrated cyanide solutions.

    Method

         Place the copper in potassium cyanide solution and leave for 10
    minutes.

    Results

         Arsenical stains dissolve in potassium cyanide solution while
    stains due to bismuth and antimony do not.

    Sensitivity

         Arsenic, about 5 mg/l.

    Quantitative assay

         Applicable to urine.

    Reagents

    1.   Silver diethyldithiocarbamate solution (5 g/l) in pyridine.

    2.   Aqueous lead acetate solution (200 g/l).

    3.   Stannous chloride (330 g/l) in aqueous hydrochloric acid
         (200 ml/l).

    4.   Concentrated hydrochloric acid (relative density 1.18).

    5.   Potassium iodide (solid).

    6.   Granulated zinc.

    Apparatus

         Modified Gutzeit apparatus (Fig. 9).

    Standards

         Dissolve 2.4 g of arsenic trichloride in 1 litre of dilute
    hydrochloric acid (1 mol/l); this gives a solution containing an
    arsenic concentration of 1 g/l. Dilute with purified water to give
    solutions containing arsenic concentrations of 0.5, 2.0, 5.0 and
    10.0 mg/l.

    Method

    1.   Clean the modified Gutzeit apparatus with acetone and dry.

    2.   Soak a plug of glass wool in lead acetate solution and allow to
         dry at room temperature.

    3.   Insert the treated glass wool into the top (capillary) end of the
         guard tube.

    4.   Introduce 3.0 ml of silver diethyldithiocarbamate solution into
         the bubbler.

    FIGURE 9

    5.   Add 2 g of potassium iodide and 50 ml of sample to the 100-ml
         conical flask, swirl until dissolved, and add 2 ml of stannous
         chloride solution and 10 ml of concentrated hydrochloric acid.

    6.   Mix well, add 10 g of granulated zinc and quickly position the
         bubbler and check the seals of all joints.

    7.   Allow the reaction to proceed for 45 minutes at room temperature.

    8.   Disconnect the bubbler and swirl gently to dissolve any complex
         formed on the walls and to mix the solution thoroughly.

    Results

         Measure the absorbance of the solution at 540 nm against a
    reagent blank (see section 4.5.2) and calculate the arsenic
    concentration using a previously prepared calibration graph. The
    calibration is linear for arsenic concentrations up to 10 mg/l.
    Germanium and antimony interfere in this assay.

    Sensitivity

         Arsenic, 0.5 mg/l.

    Clinical interpretation

         Acute ingestion of arsenical salts produces severe abdominal
    pain, vomiting and copious, bloody diarrhoea. Death from circulatory
    collapse often ensues. Inhalation of arsine produces massive
    haemolysis and renal failure. Treatment with chelating agents may be
    indicated.

    6.7  Atenolol

         2-[-4-(2-Hydroxy-3-isopropylaminopropoxy)phenyl] acetamide;
    C14H22N2O3; relative molecular mass, 266

    CHEMICAL STRUCTURE 4

    Atenolol is a cardio-selective -adrenoceptor blocking agent
    (-blocker) used in the treatment of hypertension.

         There is no simple qualitative test for atenolol, but it can be
    detected and identified by thin-layer chromatography of a basic
    solvent extract of urine, stomach contents or scene residues (see
    section 5.2.3).

    Clinical interpretation

         In acute overdose, atenolol may cause bronchoconstriction,
    hypotension and cardiac failure. Treatment is symptomatic and
    supportive, and may include the use of -agonists. Quantitative
    measurements in blood are not normally required in management.

    6.8  Atropine

         (1R,3r,5S)-Tropan-3-yl()-tropate, C17H23NO3; relative
    molecular mass 289

    CHEMICAL STRUCTURE 5

         Atropine occurs in plants such as  Atropa belladonna and
     Datura stramonium. It has potent anticholinergic activity and is
    used to reduce bronchial and salivary secretions before anaesthesia,
    to treat gastrointestinal spasm and to produce mydriasis in ophthalmic
    procedures. Atropine is also used as an antidote to poisoning with
    inhibitors of cholinesterase, such as some organophosphorus
    pesticides, carbamate pesticides and some chemical warfare agents.
    Atropine is very potent and doses of 10 mg or more can cause severe
    poisoning.

         There is no simple qualitative test for atropine, but it can be
    detected and identified by thin-layer chromatography of a basic
    solvent extract of stomach contents or scene residues (see section
    5.2.3). It may also be possible to detect atropine in urine using this
    procedure, but urinary concentrations are often very low even after
    overdosage.

    Clinical interpretation

         Acute atropine overdose may cause tachycardia, hypertension,
    pyrexia, delirium and hallucinations. Physostigmine is an effective
    antidote. Quantitative measurements in blood are of no value in
    management.

    6.9  Barbiturates

         Barbiturates are 5,5'-disubstituted derivatives of barbituric
    acid. In addition, the nitrogen atom at position 1 may be methylated
    as in methylphenobarbital, while substitution of sulfur for oxygen at
    position 2 gives thiobarbiturates such as thiopental.

         The structure of barbituric acid is shown below:

    CHEMICAL STRUCTURE 6

         Some commonly occurring barbiturates are listed in Table 18.
    Other barbiturates that may be encountered include cyclobarbital,
    cyclopentobarbital, heptabarbital, hexobarbital, methohexital and
    vinbarbital. Note that barbituric acid itself is no longer used as a
    drug.

        Table 18.  Some barbiturate hypnotics
                                                                                 

    Compound          Chemical name                                      Relative
                                                                         molecular
                                                                         mass
                                                                                 

    Amobarbital       5-Ethyl-5-isopentylbarbituric acid                 226
    Barbital          5,5-Diethylbarbituric acid                         184
    Pentobarbital     5-Ethyl-5(1-methylbutyl)barbituric acid            226
    Phenobarbital     5-Ethyl-5-phenylbarbituric acid                    232
    Secbutabarbital   5-n-Butyl-5-ethylbarbituric acid                   212
    Secobarbital      5-Allyl-5-(1-methylbutyl)barbituric acid           238
    Thiopental        5-Ethyl-5-(1-methylbutyl)-2-thiobarbituric acid    242
                                                                                 
    
         Barbiturates are potent hypnotics and sedatives, but in many
    countries only phenobarbital and (intravenous) thiopental find wide
    application nowadays. Barbiturates may also be used for euthanasia in
    veterinary medicine, and barbital sodium is used as a laboratory
    chemical, especially in buffer solutions.

         In acute poisoning it may be important to ascertain whether
    barbital or phenobarbital (so-called long-acting barbiturates), or a
    short- or medium-acting compound has been taken. This is because
    alkaline diuresis (see section 2.2.3) can enhance the excretion of
    barbital and phenobarbital, but not of other barbiturates.

         There is no reliable simple test for these compounds and a
    qualitative analysis is best performed by thin-layer chromatography of
    a solvent extract of urine, stomach contents or scene residues (see
    section 5.2.3). This should also permit identification of the type of
    barbiturate present, if not the actual compound ingested.

         The method given below will permit measurement of total
    barbiturate in a solvent extract of the specimen, and relies on the
    characteristic spectral shift shown by barbiturates on going from pH
    11 to pH 2. However, ideally a double-beam spectrophotometer is
    required (see section 4.5). Accurate measurement of individual
    barbiturates normally requires gas-liquid or high-performance liquid
    chromatography.

    Quantitative assay

         Applicable to whole blood, plasma or serum (5 ml).

    Reagents

    1.   Borate buffer, pH 8.4. Mix 22.4 g of disodium tetraborate with
         76 ml of aqueous hydrochloric acid (1 mol/l) and dilute to 2
         litres with purified water.

    2.   Aqueous hydrochloric acid (2 mol/l).

    3.   Concentrated sulfuric acid (relative density 1.83).

    4.   Concentrated ammonium hydroxide (relative density 0.88).

    5.   Sodium sulfate/charcoal mixture. Add 100 mg of activated charcoal
         to 100 g of anhydrous sodium sulfate, mix thoroughly and heat in
         an evaporating basin at 100C for 8 hours. Allow to cool and
         store in a tightly stoppered bottle.

    Standards

         Solutions containing barbital at concentrations of 5, 10, 25 and
    50 mg/l in blank human plasma, prepared by dilution from an aqueous
    solution of barbital sodium (1.12 g/l, equivalent to diethylbarbituric
    acid at a concentration of 1.00 g/l).

    Method

    1.   Add 5 ml of sample, 2 ml of hydrochloric acid and 60 ml of
         diethyl ether (with care) to a 250-ml separating funnel.

    2.   Lubricate the stopper of the funnel with purified water, insert
         and shake gently for 2 minutes.

    3.   Allow to stand for 5 minutes, and then discard the lower, aqueous
         phase through the tap of the separating funnel.

    4.   Add the diethyl ether extract to 10 ml of borate buffer in a
         second separating funnel and mix for 1 minute.

    5.   Allow to stand for 5 minutes and again discard the lower, aqueous
         phase through the funnel tap.

    6.   Wash round the funnel with 5 ml of purified water, allow to stand
         for 5 minutes and again discard the lower, aqueous phase through
         the funnel tap.

    7.   Add about 4 g of sodium sulfate/charcoal mixture to the ether
         extract in the funnel, shake to disperse, and filter the extract
         through phase-separating filter-paper into a 150-ml conical
         flask.

    8.   Add a further 20 ml of diethyl ether to the separating funnel,
         shake and add to the extract in the flask through the filter
         funnel.

    9.   Evaporate the extract to dryness on a water-bath at 40C under a
         stream of compressed air or nitrogen.

    10.  Add 5.0 ml of purified water to the dry extract in the flask,
         swirl gently and allow to stand for 5 minutes.

    11.  Filter the reconstituted extract through phase-separating filter-
         paper into a 12.5-cm test-tube.

    12.  Check the spectrophotometer zero at 240 nm using purified water
         in both sample and reference positions (1  1  4-cm fused silica
         cells, see section 4.5.2).

    13.  Add 4 ml of filtrate from the test-tube to a clean, dry cell, add
         50 l of concentrated ammonium hydroxide and mix using a plastic
         paddle. Check that the pH is about 10 (universal indicator
         paper).

    14.  Quickly measure the absorbance at 240 nm against a purified water
         blank (see section 4.5.2). If necessary, accurately dilute a
         portion of the extract with purified water to bring the reading
         on to the scale, and record the magnitude of the dilution. If a
         scanning spectrophotometer is available, scan in the region
         200-450 nm.

    15.  Repeat the reading or scan after 5 minutes.

    16.  Add 0.1 ml of concentrated sulfuric acid to the cell, mix using
         the plastic paddle, and check that the pH is about 2 (universal
         indicator paper).

    17.  Repeat the reading (240 nm) or scan (200-450 nm).

    Results

         A number of compounds can interfere. Glutethimide is hydrolysed
    rapidly at alkaline pH values, so that the absorbance at 240 nm will
    markedly decrease after 5 minutes at pH 11 (step 15 above) if this
    compound is present. The presence of other compounds, such as
    methaqualone or phenazone (e.g., dichloralphenazone), can be revealed
    by scanning in the region 200-450 nm. Addition of 0.1 ml of aqueous
    sodium hydroxide (2 mol/l) to the ammoniacal extract (step 14 above)
    produces a further characteristic spectral change (Fig. 10) which can
    be useful in qualitative work.

    FIGURE 10

         To perform a quantitative measurement, measure the difference
    between absorbance at pH 10 and at pH 2, construct a calibration graph
    by analysis of the standard barbiturate solutions, and calculate the
    barbiturate concentration in the sample.

         Alternatively, use the following formula:

    ((absorbance at pH 10) - (absorbance at pH 2))  dilution factor
                   (if any)  25 = barbiturate (mg/l)

    Sample volumes of less than 5 ml may be used, but there will be a
    corresponding loss of sensitivity unless "micro"-volume fused silica
    spectrophotometer cells are available.

    Sensitivity

         Barbiturate, 2 mg/l.

    Clinical interpretation

         Barbiturates are very toxic in overdose and may cause peripheral
    vasodilation, hypotension, shock, hypoventilation, hypothermia, coma,
    convulsions and acute renal failure. Death normally follows
    respiratory or cardiorespiratory arrest or respiratory complications.

         Plasma barbiturate concentrations greater than 10 mg/l (50 mg/l
    barbital and phenobarbital) may be associated with serious toxicity.
    Repeated oral doses of activated charcoal and/or alkaline diuresis may
    be valuable in severe poisoning with barbital and phenobarbital.
    Charcoal haemoperfusion has been used to treat severe poisoning with
    short- and medium-acting barbiturates (see section 2.2.3).

    6.10  Barium

         The most important source of barium (Ba) is barium sulfate
    (barytes, BaSO4) which is extremely insoluble in water. More soluble
    salts of barium, such as barium nitrate (BaNO3) and barium chloride
    (BaCl2), have a number of industrial uses and are relatively toxic.
    Barium sulfide (BaS) has been employed as a depilatory agent.

         There is no simple method for the measurement of barium in
    biological specimens. However, the tests described below can be used
    to indicate the presence of barium salts in stomach contents or other
    samples that contain relatively high concentrations of the element.

         The confirmatory test relies on the fact that lead sulfate is
    relatively soluble in dilute acetic acid but is precipitated in the
    presence of soluble barium salts, thus effectively enhancing the
    sensitivity of the reaction between barium and sulfate ions.

    Qualitative test

         Applicable to stomach contents and scene residues.

    Reagents

    1.   Concentrated hydrochloric acid (relative density 1.18).

    2.   Platinum wire.

    Method

    1.   Dip the end of the platinum wire in the concentrated acid.

    2.   Dip the moistened end of the wire into the test material.

    3.   Place the material in the hot part of the flame of a spirit lamp
         or microburner.

    Results

         A yellow-green flame indicates the presence of barium salts.
    Copper and thallium salts give a green flame in this test.

         If large amounts of sodium salts are present, an orange/yellow
    coloration will obscure everything else.

    Sensitivity

         Barium, 50 mg/l.

    Confirmatory test

         Applicable to stomach contents and scene residues.

    Reagents

    1.   Aqueous sulfuric acid (1 mol/l).

    2.   Aqueous lead acetate solution (100 g/l).

    3.   Aqueous acetic acid (50 ml/l).

    4.   Ammonium acetate (solid).

    Method

    1.   Mix 2 ml of lead acetate solution and 2 ml of dilute sulfuric
         acid, and add sufficient ammonium acetate to dissolve the lead
         sulfate precipitate.

    2.   Add 0.1 ml of dilute acetic acid to 1 ml of sample, add 1 ml of
         the lead sulfo-acetate solution (from step 1), and vortex-mix for
         5 seconds.

    3.   Centrifuge for 2 minutes and view the tube against a black
         background.

    Results

         A white turbidity or a white precipitate indicates the presence
    of barium. Calcium and strontium interfere in this test.

    Sensitivity

         Barium, 100 mg/l.

    Clinical interpretation

         The ingestion of soluble barium salts may produce
    gastroenteritis, ventricular fibrillation and muscular paralysis.
    Life-threatening hypokalaemia is an important feature of severe barium
    poisoning (see section 3.1.2).

    6.11  Benzodiazepines

         Most of these compounds have the general structure shown below:

    CHEMICAL STRUCTURE 7

         Some common benzodiazepines are listed in Table 19. Alprazolam,
    camazepam, clorazepate, flunitrazepam, ketazolam, loprazolam,
    lormetazepam, medazepam, midazolam, prazepam and triazolam are among
    the 60 or so members of this group.

        Table 19.  Some common benzodiazepines
                                                                                 

    Compound           Chemical name                                    Relative
                                                                        molecular
                                                                        mass
                                                                                 

    Chlordiazepoxide   7-Chloro-2-methylamino-5-phenyl-3H-              300
                       1,4-benzodiazepine 4-oxide
    Clobazam           7-Chloro-1-methyl-5-phenyl-1H-1,5-               301
                       benzodiazepin-2,4(3H,5H)-dione
    Clonazepam         5-(2-Chlorophenyl)-1,3-dihydro-7-nitro-2H-       316
                       1,4-benzodiazepin-2-one
    Diazepam           7-Chloro-1,3-dihydro-1-methyl-5-phenyl-          285
                       2H-1,4-benzodiazepin-2-one
    Flurazepam         7-Chloro-1-(2-diethylaminoethyl)-5-(2-           388
                       fluorophenyl)-1,3-dihydro-2H-1,4-
                       benzodiazepin-2-one
    Lorazepam          7-Chloro-5-(2-chlorophenyl)-1,3-dihydro-         321
                       3-hydroxy-2,H-1,4-benzodiazepin-2-one
    Nitrazepam         1,3-Dihydro-7-nitro-5-phenyl-2H-1,4-             281
                       benzodiazepin-2-one
    Oxazepam           7-Chloro-1,3-dihydro-3-hydroxy-5-                287
                       phenyl-2H-1,4-benzodiazepin-2-one
    Temazepam          7-Chloro-1,3-dihydro-3-hydroxy-1-methyl-         301
                       5-phenyl-2H-1,4-benzodiazepin-2-one
                                                                                 
    
         Benzodiazepines are used as tranquillizers, and clobazam,
    clonazepam, and diazepam are also used as anticonvulsants. Temazepam
    especially has been abused, often together with other drugs. Most
    benzodiazepines are extensively metabolized and many members of this
    group are in fact metabolites of other compounds. Thus, diazepam
    gives nordazepam, oxazepam (3-hydroxynordazepam), and temazepam
    (3-hydroxydiazepam), which are excreted in urine as glucuronide or
    sulfate conjugates.

         There is no reliable colour test for these compounds. However, on
    hydrolysis most benzodiazepines and their conjugates give rise to
    aminobenzophenones which can be extracted and analysed by thin-layer
    chromatography. Two different spray reagents are used to increase the
    discriminating power of the method,  p-dimethylaminocinnamaldehyde
    and nitrous acid/ N-(1-naphthyl)ethylenediamine (the Bratton-Marshall
    reaction).

    Qualitative test

         Applicable to urine, stomach contents and scene residues.

    Reagents

    1.   Concentrated hydrochloric acid (relative density 1.18).

    2.   Petroleum ether (40-60C boiling fraction).

    3.   Silica gel thin-layer chromatography plate (10  20 cm; 20 m
         average particle size; see section 4.4.1).

    4.   Aqueous hydrochloric acid (1 mol/l).

    5.   Toluene:glacial acetic acid (97:3).

    6.   Aqueous  p-dimethylaminocinnamaldehyde solution (5 g/l).

    7.   Aqueous trichloroacetic acid (500 g/l).

    8.   Aqueous sulfuric acid (500 ml/l).

    9.   Aqueous sodium nitrite solution (10 g/l, freshly prepared).

    10.  Aqueous ammonium sulfamate solution (50 g/l).

    11.   N-(1-Naphthyl)ethylenediamine hydrochloride (10 g/l) in
         acetone: water (4:1).

    Standards

         Flurazepam and nitrazepam, both at concentrations of 100 mg/l in
    dilute hydrochloric acid (1 mol/l).

    Method

    1.   Mix 3 ml of concentrated hydrochloric acid and 10 ml of sample or
         standard in a 30-ml glass tube with a ground-glass stopper.

    2.   Place the unstoppered tube in a boiling water-bath  in a fume
          cupboard for 30 minutes.

    3.   Cool, add 10 ml of petroleum ether, stopper the tube and rotary-
         mix for 10 minutes.

    4.   Centrifuge in a bench centrifuge for 5 minutes and transfer the
         upper, organic layer to a clean tube.

    5.   Evaporate the extract to dryness under a stream of compressed air
         or nitrogen at 60C.

    Thin-layer chromatography

    1.   Reconstitute the extract in 100 l of petroleum ether.

    2.   Divide the plate into four (two pairs of two columns), and spot
         two 25-l portions of the sample and standard extracts on to each
         pair of columns (sample extracts first, see section 4.4.2)

    3.   Develop the chromatogram (10-cm run) using toluene:acetic acid
         (saturated tank, see section 4.4.3).

    4.   Remove the plate and allow to dry.

          Take care - all of the spray reagents used are toxic. Spraying
          must be performed in a fume cupboard or under an efficient fume
          hood.

    5.   Spray one pair of columns (A) with  p-dimethylaminocinnamaldehyde
         solution followed by trichloroacetic acid.

    6.   Spray the remaining pair of columns (B) with the following
         reagents, drying between each stage: sulfuric acid, sodium
         nitrite solution, ammonium sulfamate solution and
         naphthylethylenediamine solution.

    Results

         hRf values and colour reactions of some common benzophenones are
    given in Table 20.

        Table 20.  Thin-layer chromatography of aminobenzophenones: hRf values and colour reactions
                                                                                               

    Benzophenone          Derived from:              Origin             hRf   Colour
                                                                                               
                                                                              Aa       Bb
                                                                                               

    Methylaminochloro-    Diazepam                   Diazepam           66    Pink     Purple
                                                     Ketazolam
                                                     Medazepam

                          Temazepam                  Temazepam
                                                     Diazepam
                                                     Camazepam
                                                     Medazepam
                                                     Ketazolam

    Aminochloro-          Oxazepam                   Oxazepam           40    Purple   Purple
                                                     Diazepam
                                                     Prazepam
                                                     Medazepam
                                                     Ketazolam
                                                     Chlordiazepoxide
                                                     Camazepam
                                                     Temazepam
                                                     Dipotassium
                                                     clorazepate

                          Chlordiazepoxide           Chlordiazepoxide
                          Desmethylchlordiazepoxide  Chlordiazepoxide
                          Demoxepam                  Chlordiazepoxide
                          Nordazepam                 Diazepam
                                                     Dipotassium
                                                     clorazepate
                                                     Ketazolam
                                                     Prazepam
                                                     Medazepam
                                                     Chlordiazepoxide

    Aminodichloro-        Lorazepam                  Lorazepam          45    Purple   Purple
                                                     Lormetazepam

    Aminochlorofluoro-    Desalkylflurazepam         Flurazepam         31    Purple   Purple

    Aminonitro-           Nitrazepam                 Nitrazepam         34    Pink     Purple
                                                                                               

    Table 20.  (Con't)
                                                                                               

    Benzophenone          Derived from:              Origin             hRf   Colour
                                                                                               
                                                                              Aa       Bb
                                                                                               

    Diamino-              7-Aminonitrazepam          Nitrazepam         52    Purple   Blue
                          7-Acetamidonitrazepam      Nitrazepam

    Aminonitrochloro-     Clonazepam                 Clonazepam         34    Pink     Blue
                          Loprazolam and             Loprazolam
                          metabolites
                                                                                               

    a    Visualization reagent: p-dimethylaminocinnamaldehyde solution followed by
         trichloroacetic acid.

    b    Visualization reagent: sulfuric acid followed by sodium nitrite solution,
         ammonium sulfamate solution and naphthylethylenediamine solution.
    
         Interference from other hydrolysis products can be minimized by
    extracting the petroleum ether extract (step 4) with aqueous sodium
    hydroxide (2 mol/l) on a rotary mixer for 5 minutes. Subsequently,
    separate the phases by centrifugation for 5 minutes, and evaporate the
    petroleum ether extract to dryness as described above (step 5).

         Interpretation of the results can be difficult since
    many compounds give methylaminochlorobenzophenone and/or
    aminochlorobenzophenone on hydrolysis of urine. For example, it may
    not be possible to differentiate between temazepam or oxazepam and
    diazepam or nordazepam since these compounds give a similar pattern of
    benzophenones.

         The following compounds do not themselves give benzophenones on
    hydrolysis: medazepam, triazolam, clobazam, norclobazam, and
    midazolam.

    Sensitivity

         Nordazepam (as aminochlorobenzophenone), 1 mg/l.

    Clinical interpretation

         Acute poisoning with benzodiazepines is common but, in adults,
    usually causes only drowsiness, confusion, ataxia, slurred speech,
    incoordination and sometimes coma. Respiratory depression is unusual
    in adults except with flurazepam. However, respiratory depression may
    occur if respiratory disease is already present, and in young children
    and the elderly. Benzodiazepines also have a synergistic respiratory
    depressant effect when taken with ethanol or other central nervous
    system depressants.

         Treatment is normally symptomatic and supportive, although
    flumazenil may be used as a specific antagonist (see Table 4). There
    is little need to measure plasma benzodiazepine concentrations in the
    management of acute poisoning.

    6.12  Bismuth

         Bismuth (Bi) has industrial uses in pigments and the production
    of alloys. In medicine, the main applications of bismuth salts, such
    as bismuth subsalicylate, are for the treatment of gastrointestinal
    problems like gastritis, peptic ulcer and diarrhoea. As with antimony,
    arsenic and mercury, bismuth can be detected using the Reinsch test.

    Qualitative test

         Applicable to urine, stomach contents and scene residues. Reinsch
    test - see antimony monograph (section 6.5)

    Results

         Staining on the copper can be interpreted as follows:

    purple black - antimony

    dull black - arsenic

    shiny black - bismuth

    silvery - mercury

    Selenium and tellurium may also give dark deposits, while high
    concentrations of sulfur may give a speckled appearance to the copper.

         An estimation of the concentration of bismuth in the sample can
    be made by comparison of the deposit on the copper with that obtained
    from a solution containing a known concentration of the element.

    Sensitivity

         Bismuth, about 2 mg/l.

    Confirmatory test

         Applicable to blackened copper from the test above.

    Reagents

    1.   Aqueous potassium cyanide solution (100 g/l).  Take care when
          using concentrated cyanide solutions.

    2.   Aqueous sodium sulfite solution (50 g/l, freshly prepared).

    3.   Aqueous nitric acid (3 mol/l).

    4.   Quinine/potassium iodide reagent. Dissolve 1 g of quinine sulfate
         in 100 ml of purified water containing 0.5 ml of concentrated
         nitric acid (relative density 1.42). Add 2 g of potassium iodide
         when the quinine has completely dissolved.

    Method

    1.   Place the copper in potassium cyanide solution and allow to stand
         for 10 minutes.

    2.   Wash any undissolved stain with purified water and add 1 ml of
         sodium sulfite solution and 1 ml of dilute nitric acid.

    3.   Agitate frequently for 5 minutes and add 1 ml of purified water
         followed by 1 ml of quinine/potassium iodide reagent.

    Results

         Stains due to arsenic dissolve in potassium cyanide solution,
    while stains due to antimony and bismuth do not. However, bismuth
    slowly forms an orange/brown suspension with quinine/potassium iodide
    reagent.

    Sensitivity

         Bismuth, about 2 mg/l.

    Clinical interpretation

         Acute poisoning with bismuth may cause renal damage,
    encephalopathy and peripheral neuropathy. Neurotoxicity may also occur
    after chronic treatment with bismuth salts, but is reversible if
    medication is stopped.

    6.13  Borates

         Borates are found in household products as either boric acid
    (H3BO3) or borax (sodium borate, disodium tetraborate, Na2B4O7),
    and are used in insecticides, fungicides, wood-preservatives, cleaning
    agents and water softeners. Weak solutions are used in eye-drops, eye-
    lotions, mouth washes, depilatory agents and other topical ointments.
    Small children are especially susceptible to borates and deaths have
    occurred after topical application of boric acid powder for happy
    rash. Serious borate poisoning in adults is usually the result of
    improper use. The fatal dose of boric acid or sodium borate in an
    adult is 7-35 g.

    Qualitative test

         Applicable to stomach contents and scene residues.

    Reagents

    1.   Turmeric (the spice) solution (10 g/l) in methanol.

    2.   Aqueous hydrochloric acid (1 mol/l).

    3.   Aqueous ammonium hydroxide (4 mol/l).

    Method

    1.   Soak strips (1  5 cm) of filter-paper in turmeric solution and
         allow to dry at room temperature.

    2.   Add 1 ml of dilute hydrochloric acid to 1 ml of sample and soak a
         strip of turmeric paper in the solution.

    3.   Allow the paper to dry and then moisten with ammonium hydroxide
         solution.

    Results

         A brownish-red colour is obtained initially which intensifies as
    the paper dries. A change to green-black upon moistening with ammonium
    hydroxide indicates the presence of borates. Oxidizing agents
    (including bromates, chlorates, iodates and nitrites) interfere
    because they bleach turmeric.

    Sensitivity

         Borate, 50 mg/l.

    Confirmatory test

         Applicable to stomach contents and scene residues.

    Reagent

         Carminic acid (0.5 g/l) in concentrated sulfuric acid (relative
    density 1.83).

    Method

    1.   Filter, if necessary, 5 ml of stomach contents into a 10-ml glass
         tube.

    2.   Add 0.5 ml of filtrate or scene residue to a clean tube and
         slowly add 0.5 ml of carminic acid solution down the side of the
         tube so that it forms a layer under the sample.

    Results

         A blue-violet ring at the junction of the two layers indicates
    the presence of borate. Strong oxidizing agents (including bromates,
    chlorates, iodates and nitrites) also give positive results in this
    test.

    Sensitivity

         Borate, 100 mg/l.

    Quantitative assay

         Applicable to plasma:or serum (1 ml).

    Reagents

    1.   Aqueous ammonium sulfate solution (40 g/l).

    2.   Concentrated sulfuric acid (relative density 1.83).

    3.   Carminic acid (0.2 g/l) in concentrated sulfuric acid.

    Standards

         Dissolve 0.210 g of boric acid in 100 ml of purified water
    (borate ion, 2.00 g/l) and dilute with blank serum to give standard
    solutions containing borate ion concentrations of 20, 50, 100 and
    200 mg/l.

    Method

    1.   Add 5 ml of ammonium sulfate solution to 1 ml of sample or
         standard, vortex-mix, and heat in a boiling water bath for 15
         minutes.

    2.   Centrifuge for 10 minutes and transfer the supernatant to a 10-ml
         volumetric flask.

    3.   Shake the precipitate with 2 ml of water, centrifuge as above and
         again transfer the supernatant to the flask.

    4.   Make up to 10.0 ml with purified water and mix for 5 seconds.

    5.   To 1 ml of the solution from the flask add 5 ml of concentrated
         sulfuric acid and mix thoroughly.

    6.   Add 5 ml of carminic acid solution, mix thoroughly and allow to
         stand for 10 minutes.

    7.   Read the absorbance at 600 nm against a serum blank (see section
         4.5.2).

    Results

         Plot a graph of absorbance against borate concentration in the
    calibration solutions and calculate the borate concentration in the
    sample.

    Sensitivity

         Borate, 20 mg/l.

    Clinical interpretation

         Clinical features of borate poisoning include nausea, vomiting,
    diarrhoea, coma, convulsions and circulatory collapse. Haemodialysis
    or peritoneal dialysis may be indicated in severe cases. Normally,
    serum borate concentrations range up to 7 mg/l, but serious toxicity
    may occur at concentrations of 20-150 mg/l. Death may occur at
    concentrations ranging from 200 mg/l to 1500 mg/l.

    6.14  Bromates

         Bromates, such as sodium bromate (NaBrO3), are used as
    ingredients in hair treatment (home perm) kits and are strong
    oxidizing agents. The diphenylamine test given below will detect other
    such compounds, notably chlorates, hypochlorites, iodates, nitrates
    and nitrites.

    Qualitative test

         Applicable to stomach contents and scene residues.

    Reagent

         Diphenylamine (10 g/l) in concentrated sulfuric acid (relative
    density 1.83).

    Method

    1.   Filter, if necessary, 5 ml of stomach contents into a 10-ml glass
         tube.

    2.   Add 0.5 ml of filtrate or scene residue to a clean tube and
          slowly add 0.5 ml of diphenylamine solution down the side of
         the tube so that it forms a layer under the sample.

    Results

         A positive result is indicated by a strong blue colour which
    develops immediately at the junction of the two layers. A light blue
    colour will be given by most samples of stomach contents owing to the
    presence of organic material. Since all strong oxidizing agents are
    rapidly reduced in biological samples, the test should be performed as
    soon as possible after receipt of the sample.

    Sensitivity

         Bromate, 10 mg/l.

    Confirmatory test

         Applicable to urine, stomach contents and scene residues.

    Reagents

    1.   Aqueous nitric acid (2 mol/l).

    2.   Aqueous silver nitrate solution (10 g/l).

    3.   Aqueous sodium nitrite solution (50 g/l, freshly prepared).

    4.   Concentrated ammonium hydroxide (relative density 0.88).

    Method

    1.   To 1 ml of sample add 0.2 ml of dilute nitric acid and 0.2 ml of
         silver nitrate solution and mix for 5 seconds.

    2.   If a precipitate forms (owing to the presence of halides),
         centrifuge in a bench centrifuge for 1 minute and retain the
         clear supernatant.

    3.   Add more silver nitrate solution, drop by drop, to ensure the
         complete removal of any halide, and then add 0.2 ml of sodium
         nitrite solution.

    4.   If a precipitate forms add 0.2 ml of concentrated ammonium
         hydroxide.

    Results

         A cream precipitate sparingly soluble in ammonium hydroxide
    indicates the presence of bromate. Iodate reacts similarly to halides
    in this test (see step 3).

    Sensitivity

         Bromate, 10 mg/l.

    Clinical interpretation

         Acute bromate poisoning may cause nausea, vomiting, diarrhoea,
    abdominal pain, confusion, coma and convulsions. Methaemoglobinaemia
    is often produced and this may be indicated by dark chocolate-coloured
    blood (see section 3.2.2). Blood methaemoglobin can be measured but is
    unstable, and the use of stored samples is unreliable. Treatment is
    symptomatic and supportive.

    6.15  Bromides

         Salts such as sodium bromide (NaBr) are sometimes still used as
    sedatives and anticonvulsants, and are also employed in photographic
    processing. Methyl bromide is used as a fumigant in ships' holds and
    grain silos, and is partly metabolized to bromide ion  in vivo. 
    Brominated sedatives such as carbromal also give rise to inorganic
    bromide when metabolized. The qualitative test given below serves to
    indicate the presence of inorganic bromide or iodide, and the
    appropriate confirmatory tests must then be used.

    Qualitative test

         Applicable to urine, stomach contents and scene residues.

    Reagents

    1.   Aqueous nitric acid (2 mol/l).

    2.   Aqueous silver nitrate solution (10 g/l).

    3.   Concentrated ammonium hydroxide (relative density 0.88).

    Method

    1.   Add 0.1 ml of nitric acid to 1 ml of clear test solution, mix for
         5 seconds and add 0.1 ml of silver nitrate solution.

    2.   Centrifuge to isolate any significant precipitate, decant and
         treat with 0.1 ml of concentrated ammonium hydroxide.

    Results

         A white precipitate soluble in ammonium hydroxide indicates
    chloride, an off-white precipitate sparingly soluble in ammonium
    hydroxide indicates bromide, and a creamy yellow, insoluble
    precipitate indicates iodide.

         This procedure can also be used to test for organobromine
    sedatives such as carbromal in stomach contents and scene residues.
    First boil 1 ml of the sample with 1 ml of aqueous sodium hydroxide
    (5 mol/l) for 5 minutes, cool and neutralize by slowly adding 3 ml of
    nitric acid (2 mol/l). Then proceed with step 1 above.

    Sensitivity

         Bromide, 50 mg/l.

    Confirmatory test

         Applicable to urine, stomach contents and scene residues.

    Reagents

    1.   Saturated fluorescein solution in aqueous acetic acid (600 ml/l).

    2.   Concentrated sulfuric acid (relative density 1.83).

    3.   Potassium permanganate (solid)

    Method

    1.   Soak a strip of filter-paper in fluorescein solution.

    2.   Add about 50 mg of potassium permanganate to 2 ml of test
         solution in a 10-ml test-tube.

    3.   Add 0.2 ml of concentrated sulfuric acid and hold the
         fluorescein-impregnated filter-paper in the mouth of the tube.

    Results

         Bromide is oxidized to free bromine. This reacts with the yellow
    dye fluorescein to give eosin (tetrabromofluorescein) which has a
    pink/red colour.

    Sensitivity

         Bromide, 50 mg/l.

    Quantitative assay

         Applicable to plasma or serum (2 ml).

    Reagents

    1.   Aqueous chloroauric acid. Dissolve 0.5 g of chloroauric acid
         (gold chloride, HAuCl4xH2O) in 100 ml of purified water.

    2.   Aqueous trichloroacetic acid (200 g/l).

    Standards

         Dissolve 1.29 g of sodium bromide in 500 ml of purified water
    (bromide ion 2 g/l). Prepare serial dilutions in purified water
    containing bromide ion concentrations of 0.2, 0.4, 0.6, 0.8, 1.2 and
    1.6 g/l.

    Method

    1.   Add 6 ml of trichloroacetic acid solution to 2 ml of sample in a
         10-ml test-tube, vortex-mix for 30 seconds and allow to stand for
         15 minutes.

    2.   Centrifuge in a bench centrifuge for 5 minutes and filter the
         supernatant through phase-separating filter-paper into a clean
         tube.

    3.   Add 1 ml of chloroauric acid solution to 4 ml of the clear
         supernatant and vortex-mix for 5 seconds.

    4.   Record the absorbance at 440 nm against a purified water blank
         (see section 4.5.2).

    Results

         Construct a calibration graph of bromide concentration against
    absorbance by analysis of the standard bromide solutions, and
    calculate the concentration of bromide ion in the sample. The
    calibration is linear for concentrations from 25 mg/l to 2.5 g/l. This
    method is not reliable with specimens that may give turbid
    supernatants, e.g. postmortem samples.

    Sensitivity

         Bromide, 25 mg/l.

    Clinical interpretation

         Following acute overdosage, bromides may cause nausea, vomiting
    and diarrhoea, but absorption is poor and systemic toxicity is more
    usual after chronic ingestion or exposure. In such cases, fatigue,
    irritability, anorexia, abdominal pain, skin pigmentation, visual and
    auditory hallucinations, delirium, tremor, ataxia and coma may occur.

         Normal serum bromide concentrations are less than 10 mg/l but,
    following administration of bromides in therapy, concentrations of up
    to 80 mg/l may be attained. Toxicity is usually associated with
    bromide concentrations greater than 500 mg/l. Treatment is normally
    symptomatic and supportive.

    6.16  Cadmium

         Cadmium (Cd) forms colourless salts with chemical properties
    similar to those of zinc compounds. Cadmium oxide and cadmium salts
    and alloys are used in products such as nickel-cadmium dry batteries,
    solder, paint and plastic pigments. Acute poisoning due to cadmium is
    extremely rare, but chronic toxicity has been noted after occupational
    exposure and in some instances after the diet or the water supply has
    been contaminated, as with  itai-itai (ouch-ouch) disease in Japan.

         There is no simple qualitative test for cadmium that can be
    performed on biological samples or scene residues.

    Clinical interpretation

         Chronic exposure to cadmium may lead to renal tubular damage and
    impaired lung function. Osteomalacia has also been observed in cases
    where the diet is deficient in calcium. Ingestion of cadmium salts

    causes abdominal pain, vomiting and diarrhoea, with facial oedema,
    hypotension, metabolic acidosis, depressed respiration, pulmonary
    oedema, oliguria and death in severe cases. Treatment is symptomatic
    and supportive, and may include chelation therapy.

    6.17  Caffeine

    Methyltheobromine, 7-methyltheophylline, 1,3,7-trimethylxanthine;
    C8H10N4O2; relative molecular mass, 194

    CHEMICAL STRUCTURE 8

         Caffeine is an alkaloid present in tea, coffee, cola and other
    beverages. A cup of coffee or tea may contain up to 100 mg of the
    drug. Caffeine is an ingredient of many proprietary stimulant
    preparations and is also used to treat neonatal apnoea. Metabolic
    reactions include  N-demethylation and oxidation to uric acid
    derivatives. About 85% of an oral dose is excreted unchanged in urine.
    Caffeine is an important metabolite of theophylline in neonates, and
    in adults with impaired drug handling.

         There is no simple qualitative test for caffeine, but this
    compound can be detected and identified by thin-layer chromatography
    of a basic solvent extract of urine, stomach contents or scene
    residues (see section 5.2.3). However, caffeine responds only to the
    acidified iodoplatinate visualization reagent, and sensitivity is
    poor.

    Clinical interpretation

         Acute overdosage with caffeine may cause palpitations,
    hypertension, diuresis, central nervous system stimulation, nausea,
    vomiting, marked hypokalaemia, metabolic acidosis and convulsions.
    Treatment is generally symptomatic and supportive.

    6.18  Camphor

    Bornan-2-one; C10H16O; relative molecular mass, 152

    CHEMICAL STRUCTURE 9

         Camphor is a rubefacient and is also used in mothballs. It is
    obtained by distillation from the wood of  Cinnamomum camphora or
    synthetically, and is metabolized by hydroxylation and excreted as
    glucuronides in urine. Camphor poisoning is usually due to ingestion
    of camphorated oil. The fatal dose in an adult may be as little as
    4 g.

         There is no simple qualitative test for this compound. However,
    camphor has a strong, distinctive smell and detection of this odour on
    the breath or in urine may aid the diagnosis.

    Clinical interpretation

         Ingestion of camphor may cause nausea, vomiting, headache,
    confusion, vertigo, excitement, hallucinations, tremor and dilated
    pupils. In severe cases, coma, convulsions and hepatorenal failure may
    ensure. Treatment is symptomatic and supportive.

    6.19  Carbamate pesticides

         These compounds have the general formula shown below.
    Substitution of sulfur for oxygen also occurs, but such compounds
    generally have low insecticidal activity. Some common carbamates are
    listed in Table 21.

    CHEMICAL STRUCTURE 10

        Table 21.  Some carbamate pesticides
                                                                              

    Compound      R1     R2     R3                                   Relative
                                                                     molecular
                                                                     mass
                                                                              

    Aldicarb      H      CH3    CH3SC(CH3)2CH:N                      190
    Carbaryl      H      CH3    1-naphthyl                           201
    Methiocarb    H      CH3    3,5-dimethyl-4-(methylthio)phenyl
                                                                     225
    Pirimicarb    CH3    CH3    2-dimethylamino-5,6-
                                dimethylpyrimidin-4-yl               238
    Promecarb     H      CH3    3-isopropyl-5-methylphenyl           207
    Propoxur      H      CH3    2-isopropoxyphenyl                   209
                                                                              
    
         Carbamates are widely used as insecticides, herbicides and
    fungicides. Carbamate insecticides inhibit acetylcholinesterase and
    thus evidence of exposure to such compounds can be obtained by
    measuring cholinesterase activity (see sections 3.1.5 and 6.30).
    Herbicide and fungicide carbamates, such as the dithiocarbamates, do
    not inhibit cholinesterase to any significant degree and are
    relatively nontoxic in humans. The test described here is based on a
    general reaction of carbamates with furfuraldehyde in the presence of
    hydrogen chloride.

    Qualitative test

         Applicable to stomach contents and scene residues.

    Reagents

    1.   Aqueous hydrochloric acid (2 mol/l).

    2.   Furfuraldehyde solution (100 ml/l) in methanol, freshly prepared.

    3.   Concentrated hydrochloric acid (relative density 1.18).

    Method:

    1.   Acidify 1 ml of sample with 0.5 ml of dilute hydrochloric acid
         and extract with 4 ml of chloroform on a rotary mixer for 5
         minutes.

    2.   Centrifuge in a bench centrifuge for 5 minutes, discard the
         upper, aqueous layer and filter the chloroform extract through
         phase-separating filter-paper into a clean tube.

    3.   Evaporate the extract to dryness under a stream of compressed air
         or nitrogen at 40C.

    4.   Dissolve the residue in 0.1 ml of methanol, apply a spot of the
         solution to filter-paper and allow to dry.

    5.   Apply 0.1 ml of furfuraldehyde solution to the spot, allow to dry
         and expose the paper to concentrated hydrochloric acid fumes for
         5 minutes in a fume cupboard.

    Results

         Carbamates give a black spot. Meprobamate and other non-pesticide
    carbamates interfere in this test.

    Sensitivity

         Carbamate, 100 mg/l.

    Clinical interpretation

         Exposure to carbamates may cause anorexia, abdominal pain,
    nausea, vomiting, diarrhoea, lacrimation, increased salivation,
    sweating, anxiety, ataxia and acute pulmonary oedema. Antidotal
    therapy with atropine may be indicated, but pralidoxime should not be
    used.

    6.20  Carbamazepine

    5 H-Dibenz [b,f]azepine-5-carboxamide; C15H12N2O; relative
    molecular mass, 236

    CHEMICAL STRUCTURE 11

         Carbamazepine is widely used as an anticonvulsant. Metabolic
    reactions include epoxidation to give carbamazepine-10,11-epoxide
    (which is pharmacologically active), diol formation, hydroxylation and
    conjugation. Less than 10% of a dose is excreted in urine as the
    parent compound. The estimated minimum lethal dose in an adult is 5 g.

    Qualitative test

         Applicable to stomach contents and scene residues.

    Reagents

    1.   Aqueous hydrochloric acid (2 mol/l).

    2.   Sodium hypobromite reagent. Dissolve 0.5 ml of elemental bromine
         carefully and with cooling in 5 ml of aqueous sodium hydroxide
         solution (400 g/l). Prepare freshly.

    Method

    1.   Add 1 ml of dilute hydrochloric acid to 5 ml of sample and 5 ml
         of chloroform, vortex-mix for 1 minute and centrifuge in a bench
         centrifuge for 5 minutes.

    2.   After discarding the upper, aqueous layer, add 1 ml of the
         chloroform extract to 0.2 ml of sodium hypobromite reagent in a
         clean tube and vortex-mix for 30 seconds.

    Results

         A blue-violet colour in the chloroform layer indicates the
    presence of carbamazepine. This compound and its metabolites can also
    be detected by thin-layer chromatography of an acidic extract of urine
    (see section 5.2.3).

    Sensitivity

         Carbamazepine, 250 mg/l.

    Clinical interpretation

         Carbamazepine poisoning may cause headache, dry mouth, abdominal
    discomfort, diarrhoea, constipation, ataxia, nystagmus, diplopia,
    hypotension, coma, convulsions and respiratory depression. Treatment
    is generally symptomatic and supportive.

    6.21  Carbon disulfide

         Carbon disulfide (CS2) is used as a synthetic intermediate, a
    solvent (especially in viscose rayon manufacture), a grain and soil
    fumigant, an insecticide, a corrosion inhibitor and in degreasing.
    Some 50-90% of an ingested dose of carbon disulfide is metabolized and
    excreted in urine as inorganic sulfate, thiourea, 2-mercapto-2-
    thiazolin-5-one and 2-thiothiazolidine-4-carboxylic acid (TTCA).
    Carbon disulfide has a particularly pungent smell. The ingestion of
    15 ml may prove fatal in an adult.

         There is no simple method for the detection of carbon disulfide
    in biological specimens other than by smell. However, the method given
    below can be used to assess exposure and relies on the fact that TTCA
    catalyses the decolorization of a solution of iodine by sodium azide.

    Qualitative test

         Applicable to urine.

    Reagents

    1.   Iodine-azide reagent. Dissolve 3 g of sodium azide in 25 ml of
         purified water, add 50 ml of an aqueous solution containing
         iodine (24.5 g/l) and potassium iodide (50 g/l), and dilute to
         100 ml.

    2.   Aqueous sodium dihydrogen orthophosphate (110 g/l).

    Method

    1.   Add 0.2 ml of sodium dihydrogen orthophosphate solution to 1.0 ml
         of test urine and to 1.0 ml of blank urine in separate test-
         tubes.

    2.   Vortex-mix for 2 seconds, add 20 l of iodine-azide reagent to
         each tube and again vortex-mix for 2 seconds.

    Results

         The yellow-brown colour (of iodine) is decolorized within 30
    seconds in the presence of TTCA at room temperature. It is especially
    important to analyse the blank urine as well as the test urine in this
    instance, since urine itself often has a yellow-brown colour.

    Sensitivity

         TTCA, 10 mg/l.

    Clinical interpretation

         Carbon disulfide is an excellent solvent for fat, and dermal
    contact can cause reddening, burning, cracking and peeling of the
    skin. Acute poisoning from either ingestion or inhalation may give
    rise to irritation of mucous membranes, blurred vision, headache,
    nausea, vomiting, coma, convulsions and cardiorespiratory arrest.

         Following chronic exposure, peripheral neuropathy, fatigue, sleep
    disturbance, anorexia, weight loss, depression, intellectual
    impairment, diabetes mellitus and ischaemic heart disease may occur.
    Treatment is generally symptomatic and supportive.

    6.22  Carbon monoxide

         Carbon monoxide (CO) is an important constituent of coal gas, but
    is not present in natural gas. Nowadays, common sources of carbon
    monoxide are automobile exhaust fumes, improperly maintained or
    ventilated gas or fuel oil heating systems, and smoke from all types
    of fires. Carbon monoxide is also produced  in vivo from the
    metabolism of dichloromethane.

         Carbon monoxide is highly poisonous and combines with haemoglobin
    and other haem proteins such as cytochrome oxidase, thereby limiting
    the oxygen supply to tissue and inhibiting cellular respiration. The
    affinity of carbon monoxide for haemoglobin is about 200 times that of
    oxygen. Thus, severe acute or acute-on-chronic poisoning can occur
    when relatively small quantities of carbon monoxide are present in the
    inspired air.

         The qualitative test described below is relatively insensitive
    and is useful only in the diagnosis of acute carbon monoxide
    poisoning. If a positive result is obtained then either the blood
    carboxyhaemoglobin (HbCO) or the breath carbon monoxide concentration
    should be measured without delay. The quantitative method for
    determining blood HbCO described below relies on the fact that both
    oxygenated haemoglobin and methaemoglobin (oxidized haemoglobin) can
    be reduced by sodium dithionite while HbCO is largely unaffected.

    Qualitative test

         Applicable to whole blood treated with heparin, edetic acid or
    fluoride/oxalate.

    Reagent

         Aqueous ammonium hydroxide (0.01 mol/l).

    Method

         Add 0.1 ml of blood to 2 ml of ammonium hydroxide solution and
    vortex-mix for 5 seconds.

    Results

         A pink tint in comparison with the colour obtained from a normal
    blood specimen suggests the presence of carboxyhaemoglobin. Cyanide
    may give a similar tint, but acute cyanide poisoning is generally much
    less common than carbon monoxide poisoning.

    Sensitivity

         HbCO, 20%.

    Quantitative assay

         Applicable to whole blood treated with heparin, edetic acid or
    fluoride/oxalate.

    Reagents

    1.   Aqueous ammonium hydroxide (1 ml/l).

    2.   Sodium dithionite (solid, stored in a desiccator).

    3.   A supply of pure carbon monoxide or carbon monoxide/nitrogen.

    4.   A supply of oxygen or compressed air.

    Method

    1.   Add 0.2 ml of blood to 25 ml of ammonium hydroxide solution and
         mix.

    2.   Take three approximately equal portions: x, y and z. Keep portion
         x in a stoppered tube while the following procedures are
         performed:

         (a) Saturate portion y with carbon monoxide (to give 100% HbCO)
         by bubbling the gas through the solution for 5-10 minutes. Take
         care to minimize frothing.

         (b) Saturate portion z with oxygen by bubbling pure oxygen or
         compressed air through the solution for at least 10 minutes to
         remove all bound carbon monoxide (to give 0% HbCO). Again, take
         care to minimize frothing.

    3.   Add a small amount (about 20 mg) of sodium dithionite to each
         test solution (x, y and z) and also to 10 ml of ammonium
         hydroxide solution and mix well.

    4.   Measure the absorbances of solutions x, y and z against the
         dithionite-treated ammonium hydroxide solution at 540 nm and
         579 nm.

    Results

         The percentage carboxyhaemoglobin saturation (% HbCO can be
    calculated from the equation:

            (A540/A579solution x) - (A540/A579solution z)
    %HbCO =                                                100

            (A540/A579solution y) - (A540/A579solution z)

    Approximate normal values are:

    (A540/A579 solution y) = 1.5, corresponding to 100% HbCO

    (A540/A579 solution z) = 1.1, corresponding to 0% HbCO.

    Note that the haemoglobin content of blood varies from person to
    person, and thus the volume of diluent used may need to be altered. A
    dilution giving a maximum absorbance of about 1 absorbance unit at
    540 nm is ideal.

         It is important to use sodium dithionite that has been freshly
    obtained or stored in a sealed container in a desiccator, since this
    compound is inactivated by prolonged contact with moist air.

         This method is unreliable in the presence of other pigments such
    as methaemoglobin (indicated by a relatively high absorbance in the
    region 580-600 nm, see Fig. 11). Lipaemic blood specimens may give
    turbid suspensions which also give unreliable results.

         The measurements are performed at the point of maximum difference
    of absorbance (540 nm, lambdamax HbCO) and the point of equal
    absorbance (579 nm, isobestic point). The reading at 579 nm is taken
    on a very steep slope (Fig. 11), and the wavelength is critical.
    Spectrophotometers with a relatively broad band-pass (4-5 nm) should
    not be used, since it will be impossible to perform the measurement
    with the accuracy required. Even if an instrument with a narrow band-
    pass is available, it is important to ensure that it is accurately
    calibrated, although the effect of minor variations can be minimized
    by using the following procedure:

    FIGURE 11

    1.   Measure the absorbance of solution z (0% HbCO) against the
         dithionite-treated ammonium hydroxide solution at 540 nm. If the
         ratio (A540/A579) for 0% HbCO is assumed to be 1.1, the
         absorbance of this solution at 579 nm can be calculated.

    2.   Adjust the wavelength setting of the instrument to give this
         reading if not already attained at 579 nm. Alternatively, the
         spectra from the three solutions can be recorded using a scanning
         spectrophotometer, if available, and the measurements performed
         directly. Examples of the spectra that should be obtained are
         given in Figure 11. The presence of the twin absorption peaks
         ("rabbit's ears") is a useful qualitative feature.

    Sensitivity

         HbCO, approximately 10%.

    Clinical interpretation

         Features of acute carbon monoxide poisoning include headache,
    nausea, vomiting, haematemesis, hyperventilation, cardiac arrhythmias,
    pulmonary oedema, coma and acute renal failure. Cyanosis is commonly
    absent, so that skin and mucosae remain pink even in the presence of
    severe tissue hypoxia. Death often ensues from respiratory failure.
    Late neuropsychiatric sequelae are an increasingly recognized
    complication.

         Treatment consists of removal from the contaminated atmosphere
    and administration of 100% oxygen via a well-fitting face-mask.
    Hyperbaric oxygen may be indicated in certain cases, and is especially
    effective in preventing the development of late sequelae, but
    facilities where this can be given are rare.

         Once the patient is removed from the contaminated atmosphere,
    carboxyhaemoglobin is dissociated rapidly, especially if oxygen is
    administered in treatment. HbCO measurements are therefore often
    unhelpful as an indication of the severity of poisoning except in
    forensic toxicology. A simple guide to the interpretation of blood
    HbCO results is given in Table 22.

    Table 22.  Interpretation of blood carboxyhaemoglobin (HbCO) results
                                                                        

    HbCO saturation     Associated with:
          (%)
                                                                        

          3-8           Cigarette smokers
         < 15           Heavy smokers (30-50 cigarettes per day)
          20            Danger to heart disease patients
         20-50          Progressive loss of mental and physical
                        coordination resembling ethanol intoxication
         > 50           Coma, convulsions, cardiorespiratory arrest, death
                                                                        

    6.23  Carbon tetrachloride

    Tetrachloromethane; CCl4; relative molecular mass, 154

         Carbon tetrachloride was widely used as a dry-cleaning and
    degreasing agent and in fire extinguishers. However, as with
    chloroform, exposure to carbon tetrachloride frequently gives rise to
    hepatorenal damage and nowadays usage is largely restricted to
    fumigation of grain and industrial applications.

         Massive exposure to carbon tetrachloride may be detectable in
    urine using the Fujiwara test, possibly because chloroform is a minor
    metabolite or contaminant; carbon tetrachloride itself does not react
    in this test.

    Qualitative test

         Applicable to urine. Fujiwara test.  This test must be performed
     in a fume cupboard.

    Reagents

    1.   Aqueous sodium hydroxide solution (5 mol/l, i.e., 200 g/l).

    2.   Aqueous trichloroacetic acid (10 mg/l).

    Method

    1.   To separate 10-ml tubes add 1-ml portions of:

         (a) test urine;

         (b) purified water; and

         (c) trichloroacetic acid solution.

    2.   Add 1 ml of sodium hydroxide solution and 1 ml of pyridine to
         each tube, mix gently and fit with a loose stopper.

    3.   Heat in a boiling water-bath for 2 minutes.

    Results

         An intense red/purple colour in the upper, pyridine layer
    indicates the presence of trichloro compounds. The blank analysis
    excludes contamination with compounds such as chloroform from the
    laboratory atmosphere. Compounds such as chloral hydrate,
    dichloralphenazone and trichloroethylene, which are extensively
    metabolized to trichloroacetic acid, give strong positive results in
    this test.

    Sensitivity

         Trichloroacetate, 1 mg/l.

    Clinical interpretation

         Acute poisoning with carbon tetrachloride is rare. Clinical
    features include ataxia, nausea, vomiting, coma, convulsions,
    respiratory depression and cardiac arrhythmias. Hepatic and renal
    damage commonly occur. Treatment is symptomatic and supportive,
    although acetylcysteine may protect against liver and kidney damage.

    6.24  Chloral hydrate

    Chloral; 2,2,2-trichloroethane-1,1-diol; C2H3O2Cl3; relative
    molecular mass, 165

         Cl  OH
         '   '
         '   '
    Cl---C---C---H
         '   '
         '   '
         Cl  OH

         Chloral hydrate is a mild sedative and hypnotic agent. The
    pharmacological activity of chloral, and of the related compound
    dichloralphenazone, is thought to derive largely from a metabolite,
    2,2,2-trichloroethanol, which is in turn metabolized to
    trichloroacetic acid. This latter compound can be detected in urine
    using the Fujiwara test.

    Qualitative test

         Applicable to urine. Fujiwara test - see carbon tetrachloride
    monograph (section 6.23).  This test must be performed in a fume
     cupboard.

    Results

         An intense red/purple colour in the upper, pyridine layer
    indicates the presence of trichloro compounds. The blank (purified
    water) analysis excludes contamination with compounds such as
    chloroform from the laboratory atmosphere. Other trichloro compounds
    react in this test, but trichloroacetic acid is by far the most common
    compound encountered.

         This test is very sensitive and will detect a therapeutic dose of
    chloral hydrate 12-24 hours after ingestion. However, other compounds,
    notably the solvent trichloroethylene, also give rise to
    trichloroacetic acid  in vivo and caution must be exercised in
    reporting results.

    Sensitivity

         Trichloroacetate, 1 mg/l.

    Clinical interpretation

         Acute poisoning with chloral hydrate can cause vomiting,
    excitement, ataxia, confusion, drowsiness, stupor, hypotension, coma,
    cardiac arrhythmias, respiratory depression and pulmonary oedema.
    Treatment is normally symptomatic and supportive.

    6.25  Chloralose

    alpha-Chloralose;  (R)-1,2- O-(2,2,2-trichloroethylidene)-alpha-D-
    glucofuranose; C8H11Cl3O6; relative molecular mass, 310

    CHEMICAL STRUCTURE 12

         Chloralose is a hypnotic drug and has been used as a surgical
    anaesthetic in laboratory animals. It is also used as a bird repellent
    on seed grain and as a rodenticide, especially against mice, in cooler
    climates. The dose associated with toxicity in adults is about 1 g.

         Chloralose may be oxidized with periodic acid to trichloroacetic
    acid, which can be detected using the Fujiwara test as for carbon
    tetrachloride. It was thought that chloralose undergoes hydrolysis
     in vivo, and that urine from patients who had ingested chloralose
    would give a positive reaction without periodate oxidation. However,
    recent work suggests that this is not the case.

    Qualitative test

         Applicable to plasma or serum, urine, stomach contents and scene
    residues.  This test must be performed in a fume cupboard.

    Reagents

    1.   Periodic acid reagent. Mix 3 g of sodium periodate and 3 ml of
         aqueous sulfuric acid (0.5 mol/l) and dilute to 100 ml with
         water.

    2.   Aqueous sodium hydroxide solution (5 mol/l, i.e., 200 g/l).

    3.   Aqueous trichloroacetic acid (10 mg/l).

    Method

    1.   Add 1 ml of periodic acid reagent to 1 ml of test solution (or a
         portion of a solid residue extracted with 1 ml of water) in a
         10-ml glass test-tube.

    2.   Mix and allow to stand for 5 minutes.

    3.   To separate 10-ml tubes add 2-ml portions of:

         (a) test solution;

         (b) purified water;

         (c) trichloroacetic acid solution.

    4.   Add 1 ml of sodium hydroxide solution and 1 ml of pyridine to all
         four tubes, mix gently and fit with a loose stopper.

    5.   Heat in a boiling water-bath for 2 minutes.

    Results

         An intense red/purple colour in the upper, pyridine layer of the
    periodate-treated tube indicates the presence of chloralose. The
    sample analysis without periodate is to exclude the presence of
    compounds, such as chloral hydrate, that give rise to trichloroacetic
    acid  in vivo. The blank analysis excludes contamination with
    chloroform from the laboratory atmosphere.

    Sensitivity

         Trichloroacetate, 1 mg/l.

    Clinical interpretation

         Ingestion of chloralose may cause drowsiness, hypotonia and coma.
    Treatment is generally symptomatic and supportive.

    6.26  Chlorates

         Sodium chlorate (NaClO3) is used as a weedkiller and in matches
    and fireworks. Chlorates are also used in small amounts in throat
    gargles and toothpastes. In an adult, serious poisoning may follow the
    ingestion of 15 g of sodium chlorate. Chlorates are strong oxidizing
    agents, and the test given below will also detect compounds with
    similar properties, such as bromates, hypochlorites, iodates, nitrates
    and nitrites.

    Qualitative test

         Applicable to stomach contents and scene residues.

    Reagent

         Diphenylamine (10 g/l) in concentrated sulfuric acid (relative
    density 1.83).

    Method

    1.   Filter 5 ml of stomach contents into a 10-ml glass tube.

    2.   Add 0.5 ml of filtrate or scene residue to a clean tube and
          slowly add 0.5 ml of diphenylamine solution down the side of
         the tube so that it forms a layer under the sample.

    Results

         A true positive is indicated by a strong blue colour which
    develops immediately at the junction of the two layers. A light blue
    colour will be given by most samples of stomach contents owing to the
    presence of organic material. Since all strong oxidizing agents are
    reduced rapidly in biological samples, the test should be performed as
    soon as possible after receipt of the sample.

    Sensitivity

         Chlorate, 10 mg/l.

    Confirmatory test

         Applicable to stomach contents and scene residues.

    Reagents

    1.   Manganous sulfate reagent. Mix saturated aqueous manganous
         sulfate with  o-phosphoric acid (1:1).

    2.   Diphenylcarbazide (10 g/l) in methanol.

    Method

    1.   To 0.1 ml of test solution add 0.2 ml of manganous sulfate
         reagent and warm briefly over a spirit lamp or microburner.

    2.   Cool and add 0.1 ml of diphenylcarbazide solution.

    Results

         A purple/violet colour, which is intensified after cooling and
    adding diphenylcarbazide, indicates chlorate.

         Persulfates and periodates give a similar reaction; persulfates
    can be eliminated by evaporating the test solution with 0.1 ml of
    concentrated sulfuric acid (relative density 1.83) and 0.1 ml of
    aqueous silver nitrate (10 g/l).

    Sensitivity

         Chlorate, 100 mg/l.

    Clinical interpretation

         Acute poisoning with chlorates may cause nausea, vomiting,
    diarrhoea, abdominal pain, confusion, coma and convulsions.
    Methaemoglobinaemia is often produced and this may be indicated by
    dark chocolate-coloured blood (see section 3.2.2). Blood
    methaemoglobin can be measured but is unstable, and the use of stored
    samples is unreliable. Treatment is symptomatic and supportive.

    6.27  Chloroform

    Trichloromethane; CHCl3; relative molecular mass, 119

         Chloroform was used as an anaesthetic and general solvent, but is
    relatively toxic since it is partly metabolized to phosgene (COCl2)
    which is a potent hepatorenal toxin.

         Chloroform can be detected readily using the Fujiwara test.
    However, this test will also detect ingestion or exposure to compounds
    that are extensively metabolized to trichloroacetic acid, such as
    chloral hydrate, dichloralphenazone and trichloroethylene.

    Qualitative test

         Applicable to urine. Fujiwara test - see carbon tetrachloride
    monograph (section 6.23).  This test must be performed in a fume
     cupboard.

    Results

         An intense red/purple colour in the upper, pyridine layer
    indicates the presence of trichloro compounds. The blank analysis
    excludes contamination with compounds such as chloroform from the
    laboratory atmosphere. Trichloroacetic acid is by far the most common
    compound encountered in this test.

    Sensitivity

         Trichloroacetate, 1 mg/l.

    Clinical interpretation

         Acute poisoning with chloroform is rare. Clinical features
    include ataxia, nausea, vomiting, coma, convulsions, respiratory
    depression, cardiac arrhythmias and hepatorenal damage. Treatment is
    symptomatic and supportive. Acetylcysteine may protect against
    hepatorenal damage (see Table 4).

    6.28  Chlorophenoxy herbicides

         These compounds have the general formula shown below. Some common
    chlorophenoxy herbicides are listed in Table 23.

    CHEMICAL STRUCTURE 13

         2,4-D (not to be confused with DNOC, i.e. dinitro- o-cresol, see
    section 6.42) and related compounds are used to control broad-leaved
    weeds in lawns and in cereal crops and, at higher application rates,
    for total vegetation control. They are frequently encountered as
    mixtures, both with other members of the group and with other
    pesticides.

    Qualitative test

         Applicable to stomach contents and scene residues.

    Reagents

    1.   Aqueous hydrochloric acid (1 mol/l).

    2.   Sodium nitrite (100 g/l) in concentrated sulfuric acid (relative
         density 1.83), freshly prepared.  Take care - brown nitrogen
          dioxide fumes may be evolved.

    3.   Chromotropic acid (2,5-dihydroxynaphthalene-2,7-disulfonic acid)
         (2 g/l) in concentrated sulfuric acid (relative density 1.83).

        Table 23.  Some chlorophenoxy herbicides
                                                                                                      

    Compound         Chemical name                        R1     R2     R3                 Relative
                                                                                           molecular
                                                                                           mass
                                                                                                      

    2,4-D            2,4-Dichlorophenoxyacetic acid       Cl     H      CH2COOH            221
    2,4-DP           2-(2,4-Dichlorophenoxy)-             Cl     H      CH(CH3)COOH        235
     (dichlorprop)     propionic acid
    MCPA             4-Chloro-2-methylphenoxyacetic       CH3    H      CH2COOH            201
                       acid
    MCPP             2-(4-Chloro-2-methylphenoxy)-        CH3    H      CH(CH3)COOH        215
     (mecoprop)        propionic acid
    2,4,5-T          2,4,5-Trichlorophenoxyacetic         Cl     Cl     CH2COOH            256
                       acid
    2,4,5-TP         2-(2,4,5-Trichlorophenoxy)-          Cl     Cl     CH(CH3)COOH        270
     (fenoprop)        propionic acid
                                                                                                      
    
    Method

    1.   Add 1 ml of dilute hydrochloric acid to 10 ml of sample, and
         extract with 20 ml of toluene on a rotary mixer for 5 minutes.

    2.   Centrifuge for 5 minutes, remove the upper, toluene layer, and
         extract the residue with a second 20-ml portion of toluene.

    3.   Combine the toluene extracts and evaporate to dryness under a
         stream of compressed air or nitrogen in a water-bath at 60C.

    4.   Dissolve the residue in 0.2 ml of concentrated sulfuric acid and
         divide between two wells of a porcelain spotting tile.

    5.   Add 0.1 ml of sodium nitrite solution to one well and 0.1 ml of
         chromotropic acid solution to the other.

    6.   Heat the tile in a beaker over a boiling water-bath or on a hot
         plate at 80C.

    Results

         The colours given by some common chlorophenoxy compounds are
    given in Table 24. These tests are not specific and can only be used
    to indicate the presence of chlorophenoxy compounds.

    Table 24.  Colour reactions of some chlorophenoxy compounds
                                                                

    Compounda     With sodium nitrite     With chromotropic acid
                                                                

    2,4-D         Brown                   Purple
    2,4,-DP       Dark brown              Light purple
    MCPA          Light brown             Light purple
    MCPP          Light brown             Purple
    2,4,5-T       No reaction             Purple
    2,4,5-TP      No reaction             Light pink/purple
                                                                

    a    For full chemical names see Table 23.

    Sensitivity

         Chlorophenoxy compounds, 500 mg/l.

    Clinical interpretation

         Absorption of chlorophenoxy herbicides may lead to vomiting,
    diarrhoea, areflexia, muscle weakness, pulmonary oedema and coma, with
    death in severe cases. Alkalinization may increase the renal excretion
    of 2,4-D and other chlorophenoxy compounds, and also protect against
    systemic toxicity (see section 2.2.3).

    6.29  Chloroquine

    7-Chloro-4-(4-diethylamino-1-methylbutylamino)quinoline; C18H26ClN3;
    relative molecular mass, 320

    CHEMICAL STRUCTURE 14

         Chloroquine is a derivative of 4-aminoquinoline and is commonly
    used to treat malaria. Chloroquine has a long half-life in the body
    (25-60 days) and several metabolic products are formed, initially by
     N-dealkylation and deamination. As little as 1 g of chloroquine may
    cause death in a young child, and fatalities in adults have occurred
    after the ingestion of between 3 and 44 g.

         There is no simple test for chloroquine in biological fluids, but
    this compound and its metabolites can be detected by thin-layer
    chromatography of a basic extract of urine (see section 5.2.3). Like
    quinine, chloroquine fluoresces under ultraviolet light (254 nm and
    366 nm), and this provides an additional feature to aid
    identification.

    Clinical interpretation

         Acute chloroquine poisoning can develop within 30 minutes of
    ingestion. Clinical features include nausea, vomiting, abdominal pain,
    diarrhoea, tinnitus, blurred vision, dizziness, agitation,
    hypotension, coma, convulsions and respiratory depression. Sudden
    cardiorespiratory arrest may occur in severe cases. Treatment is
    generally symptomatic and supportive, but the specific combination of
    diazepam and epinephrine has proved particularly effective.

    6.30  Cholinesterase activity

         Many insecticides, such as carbamate and organophosphorus
    compounds, interfere with nerve transmission by inhibiting
    acetylcholinesterase. Semi-quantitative measurement of plasma
    cholinesterase activity provides a simple method of assessing exposure
    to these compounds (see section 3.1.5).

    Qualitative test

         Applicable to plasma or serum.

    Reagents

    1.   Dithiobisnitrobenzoate reagent. 5,5'-Dithiobis(2-nitrobenzoic
         add) (0.2 g/l) in sodium dihydrogen orthophosphate buffer
         (0.1 mol/l, pH 7.4).

    2.   Aqueous acetylthiocholine iodide solution (5 g/l).

    3.   Aqueous pralidoxime chloride solution (200 g/l).

    4.   Plasma or serum from an unexposed individual (control plasma).

    Method

    1.   Add 2.0 ml of dithiobisnitrobenzoate reagent and 1.0 ml of
         acetylthiocholine iodide solution to each of three 10-ml test-
         tubes.

    2.   Add 20 l of control plasma to one tube and 20 l of test plasma
         to a second.

    3.   Add 20 l of pralidoxime solution and 20 l of test plasma to the
         third tube.

    4.   Vortex-mix the contents of all three tubes and allow to stand at
         room temperature for 2 minutes.

    Results

         The presence of an acetylcholinesterase inhibitor is indicated if
    the yellow colour in the control tube is deeper than in the test tube.
    If the colour in the tube containing pralidoxime is similar to that in
    the control tube, this provides further confirmation that an inhibitor
    of acetylcholinesterase is present in the sample (see section 3.1.5).

    Clinical interpretation

         Exposure to organophosphorus pesticides may cause bronchorrhoea,
    respiratory distress, excessive salivation, nausea, muscle weakness
    and eventually paralysis. Treatment is supportive, but should also
    include the administration of atropine and pralidoxime.

         Exposure to carbamate pesticides may cause anorexia, abdominal
    pain, nausea, vomiting, diarrhoea, lacrimation, increased salivation,
    sweating, anxiety, ataxia and acute pulmonary oedema. Antidotal
    therapy with atropine may be indicated, but pralidoxime should not be
    used.

    6.31  Clomethiazole

    Chlormethiazole; 5-(2-chloroethyl)-4-methylthiazole; C6H8ClNS;
    relative molecular mass, 162

    CHEMICAL STRUCTURE 15

         Clomethiazole is used as a hypnotic in elderly patients, as an
    anticonvulsant, and in the treatment of alcohol dependence and drug
    withdrawal. Less than 5% of an oral dose is excreted unchanged in
    urine, and a large number of metabolites have been identified.
    Clomethiazole has a characteristic smell on the breath and in stomach
    contents.

         There is no simple qualitative test for clomethiazole, but this
    compound and its metabolites can be detected and identified by thin-
    layer chromatography of a basic solvent extract of urine (see section
    5.2.3).

    Clinical interpretation

         Acute poisoning with clomethiazole may cause sneezing, increased
    salivation, conjunctival irritation, hypotension, hypothermia, coma
    and respiratory depression. Ethanol potentiates the depressant effects
    of clomethiazole on the central nervous system, and these compounds
    are often encountered together in fatal cases. Treatment is
    symptomatic and supportive.

    6.32  Cocaine

    Methyl benzoylecgonine; (1 R,2 R,3 s,5 S)-2-methoxycarbonyltropan-
    3-yl benzoate; C17H21NO4; relative molecular mass, 303

    CHEMICAL STRUCTURE 16

         Cocaine is an alkaloid obtained from coca, the dried leaves of
     Erythroxylon coca and other species of  Erythroxylon, or by
    synthesis from ecgonine. The hydrochloride salt is an effective local
    anaesthetic when used at concentrations of 10-200 g/l, but is normally
    only applied topically because of the risk of systemic toxicity if
    given by other routes.

         Cocaine is frequently abused by injection or inhalation
    (sniffing, snorting) into the nasal passages; ingested cocaine has
    less effect owing to hydrolysis in the gastrointestinal tract. Cocaine
    free-base (crack) is very rapidly absorbed when inhaled into the nasal
    passages or smoked. The estimated minimum fatal dose in an adult is
    1-2 g, but addicts may tolerate up to 5 g/day. The principal
    metabolites are benzoylecgonine, ecgonine and ecgonine methyl ester.
    Only 1-9% of an intravenous dose is excreted in urine as cocaine,
    while 35-55% is excreted as benzoylecgonine.

         There is no simple qualitative test for cocaine, but this
    compound and its metabolites can be detected and identified by thin-
    layer chromatography of a basic solvent extract of urine (see section
    5.2.3).

    Clinical interpretation

         Acute cocaine poisoning may cause euphoria, restlessness,
    vomiting, pyrexia, mydriasis, delirium, tremor, hyperreflexia,
    hypertension, hyperventilation, convulsions and cardiorespiratory
    failure. Treatment is symptomatic and supportive.

    6.33  Codeine

    Morphine methyl ether; 3- O-methylmorphine monohydrate;
    C18H21NO3H2O; relative molecular mass, 317

    CHEMICAL STRUCTURE 17

         Codeine is a narcotic analgesic obtained either from opium or by
    methylation of morphine. Codeine is metabolized by  O-demethylation
    and  N-demethylation to give morphine and norcodeine, respectively,
    and by conjugation to form glucuronides and sulfates of both parent
    drug and metabolites. The estimated fatal dose of codeine in an adult
    is 800 mg. However, codeine is much less toxic than morphine, and
    death directly attributable to codeine is rare.

         There is no simple qualitative test for codeine, but this
    compound and norcodeine can be detected and identified by thin-layer
    chromatography of a basic solvent extract of urine (see section
    5.2.3).

    Clinical interpretation

         Acute overdosage with codeine gives rise to pinpoint pupils,
    hypotension, hypothermia, coma, convulsions, pulmonary oedema and
    cardiac arrhythmias. Death may ensue from profound respiratory
    depression. Naloxone rapidly reverses the central toxic effects of
    codeine (see section 2.2.2).

    6.34  Copper

         Copper salts such as copper(II) sulfate, chloride, and carbonate
    and cuprammonium salts such as cuprammonium carbonate (Cu(NH3)2CO3)
    are used as insecticides and fungicides. As with iron salts, copper
    salts often impart a blue or green colour to stomach contents.
    Cuprammonium salts are much more toxic than copper salts owing to
    rapid absorption and the intrinsic toxicity of the cuprammonium ion.
    Acute poisoning may also follow inhalation of metallic copper fumes or
    powder.

    Qualitative test

         Applicable to stomach contents and scene residues.

    Reagents

    1.   Dithiooxamide in methanol (10 g/l).

    2.   Concentrated ammonium hydroxide (relative density 0.88).

    Method

    1.   Slowly place 0.1 ml of sample on a filter-paper to give a spot no
         greater than 1 cm in diameter, drying with a hairdrier if
         necessary.

    2.   Expose the spot to ammonia fumes from concentrated ammonium
         hydroxide  in a fume cupboard, and add 0.1 ml of dithiooxamide
         solution to the spot.

    Results

         Copper salts give an olive-green stain. Chromium salts also give
    a green stain, which is normally visible before the dithiooxamide is
    added. A number of other metals give yellow-brown or red-brown colours
    with this reagent.

    Sensitivity

         Copper, 1 mg/l.

    Confirmatory test

         Applicable to stomach contents and scene residues.

    Reagents

    1.   Aqueous zinc acetate solution (10 g/l).

    2.   Ammonium mercurithiocyanate reagent. Mix 8 g of mercuric chloride
         and 9 g of ammonium thiocyanate in 100 ml of purified water.

    3.   Aqueous hydrochloric acid (0.01 mol/l).

    Method

    1.   Place 0.1 ml of sample in a well of a porcelain spotting tile and
         add 0.05 ml of dilute hydrochloric acid.

    2.   Mix 0.1 ml of ammonium mercurithiocyanate reagent with 0.1 ml of
         zinc acetate solution and add to the sample in the well.

    Result

         A violet precipitate of zinc mercurithiocyanate forms in the
    presence of copper salts.

    Sensitivity

         Copper, 50 mg/l.

    Quantitative assay

         Applicable to plasma or serum (1 ml).

    Reagents

    1.   Oxalyl dihydrazide reagent. Mix 8 ml of saturated aqueous oxalyl
         dihydrazide, 12 ml of concentrated ammonium hydroxide (relative
         density 0.88), 20 ml of aqueous acetaldehyde (400 ml/l) and 20 ml
         of purified water.

    2.   Aqueous trichloroacetic acid (200 g/l).

    3.   Aqueous hydrochloric acid (2 mol/l).

    Standards

         Dissolve 1.00 g of copper foil, mesh or wire in a minimum volume
    of nitric acid (500 ml/l) and make up to 1 litre with dilute nitric
    acid (10 ml/l). Dilute portions of this solution with water to give
    solutions containing copper ion concentrations of 1.0, 2.0 and
    5.0 mg/l.

    Method

    1.   Add 0.7 ml of dilute hydrochloric acid to 1 ml of sample or
         standard in a plastic centrifuge tube, mix and allow to stand for
         15 minutes.

    2.   Add 1 ml of trichloroacetic acid solution and mix thoroughly.

    3.   Allow to stand for 15 minutes and then centrifuge for 5 minutes.

    4.   Add 3 ml of oxalyl dihydrazide reagent to 1 ml of supernatant,
         mix and allow to stand for 20 minutes.

    Results

         Read the absorbance of the solution at 542 nm against an aqueous
    blank (see section 4.5.2) carried through the procedure. Plot the
    absorbance of the standard solutions against copper concentration, and
    calculate the copper concentration in the sample. The calibration
    graph is linear for copper concentrations of 1-25 mg/l.

    Sensitivity

         Copper, 1 mg/l.

    Clinical interpretation

         Ingestion of copper or cuprammonium salts leads initially to
    gastrointestinal symptoms (metallic taste, nausea, vomiting,
    epigastric pain and diarrhoea). In severe cases, hepatic damage
    (particularly in children), renal damage, haemolysis, coma and
    circulatory collapse may ensue. Normal serum copper concentrations are
    0.7-1.6 mg/l, but in severe acute poisoning concentrations greater
    than 5 mg/l may be attained. Treatment is symptomatic and supportive,
    but may also include chelation therapy.

    6.35  Coumarin anticoagulants

    Phenprocoumon (4-hydroxy-3-(1-phenylpropyl)coumarin; C18H16O3;
    relative molecular mass, 280) and warfarin (4-hydroxy-3-(3-oxo-1-
    phenylbutyl)coumarin; C19H16O4; relative molecular mass, 308) are
    substituted 4-hydroxycoumarins.

    CHEMICAL STRUCTURE 18

         These compounds are widely used therapeutic agents; warfarin is
    also used as a rodenticide. Both inhibit blood coagulation by
    interfering with the synthesis of vitamin-K-dependent clotting
    factors. Their action is cumulative so that toxicity normally results
    from chronic administration. In contrast, severe toxicity may occur
    following a single large dose of a "superwarfarin" rodenticide such as
    difenacoum or brodifacoum.

         The prothrombin time (see section 3.2.1) provides a simple, but
    nonspecific, means of measuring the severity of acute anticoagulant
    poisoning and of monitoring treatment. The simple method given below
    can be used to assess plasma phenprocoumon and warfarin
    concentrations.

    Qualitative test

         Applicable to plasma or serum (1.0 ml).

    Reagents

    1.   Aqueous hydrochloric acid (1 mol/l).

    2.    n-Butyl acetate:chloroform:aqueous formic acid (850 ml/l)
         (60:40: 10).

    3.   Triethylamine (50 g/l) in  n-hexane.

    4.   Silica gel thin-layer chromatography plate (20  20 cm, 20 m
         average particle size; see section 4.4.1).

    Standards

         Serum containing phenprocoumon or warfarin concentrations of 0,
    1, 5 and 10 mg/l.

    Method

    1.   To 1.0 ml of sample or standard add 0.9 ml of dilute hydrochloric
         acid, 0.1 ml of acetone and 5 ml of chloroform.

    2.   Mix for 2 minutes on a mechanical shaker and then centrifuge for
         10 minutes.

    3.   Remove the upper (aqueous) layer, filter the extract through
         phase-separating filter-paper and evaporate to dryness under a
         stream of compressed air or nitrogen.

    Thin-layer chromatography

    1.   Dissolve the residues in 50 l of chloroform, spot on the plate,
         and develop (10-cm run) in  n-butyl acetate:chloroform:formic
         acid (saturated tank; see section 4.4.3).

    2.   Allow the solvent to evaporate completely, develop again in
         triethylamine: n-hexane, and inspect under ultraviolet light
         (366 nm).

    Results

         Warfarin (hRf about 87) shows a dark purple fluorescence,
    phenprocoumon (hRf about 95) a brighter purple. The plasma
    concentrations of either compound can be assessed by comparison with
    the results obtained from the standards.

    Sensitivity

         Warfarin or phenprocoumon, 0.5 mg/l.

    Clinical interpretation

         Features of acute poisoning with anticoagulants include the
    occurrence of petechiae, spontaneous bruising, haematoma formation
    and frank haemorrhage, especially from the genitourinary and
    gastrointestinal tracts. Serum concentrations of either compound
    greater than 5 mg/l are often accompanied by haemorrhagic
    complications. Phenprocoumon and warfarin have long plasma half-lives
    (6-7 days and 0.5-3 days, respectively), and patients with high serum
    concentrations should be treated promptly. Therapy consists of vitamin
    K supplementation until a prothrombin time in the normal range is
    obtained. In very serious cases, intravenous administration of fresh
    frozen plasma or purified clotting factors may be considered.

    6.36  Cyanide

         Cyanide (CN-) poisoning may be encountered after the inhalation
    of hydrogen cyanide (HCN) or after the ingestion of hydrocyanic acid
    or potassium or sodium cyanide. Complex cyanide solutions are used in
    metal electroplating and acidification of such solutions often leads
    to the release of hydrogen cyanide. Cyanogenic glycosides and other
    nitrile-containing compounds, such as amygdalin, which release cyanide
     in vivo occur in a number of plant tissues, including peach and
    apricot kernels, cassava root and lima beans.

         Thiocyanate insecticides (ethyl thiocyanate, methyl thiocyanate)
    are also metabolized to cyanide ion  in vivo and can cause serious
    toxicity. Cyanide is also a metabolite of sodium nitroprusside (used
    as a vasodilator) and some other nitrile-containing compounds, but
    cyanide poisoning is unusual in such cases. Inorganic thiocyanates
    and ferricyanide and ferrocyanide salts do not give rise to cyanide
     in vivo and are relatively nontoxic.

         The qualitative test described below is based on the formation of
    a blue ferriferrocyanide complex (Prussian blue) with ferrous ions.
    Two microdiffusion methods (section 4.3.3) applicable to blood
    specimens are also given, both based on the liberation of hydrogen
    cyanide and subsequent formation of a coloured complex. The first, the
     p-nitrobenzaldehyde/ o-dinitrobenzene method, can be used to give a
    rapid semiquantitative result, while the pyridine/barbituric acid
    method should be used for a full quantitative analysis.

    Qualitative test

         Applicable to stomach contents and scene residues.  Take
     care - specimens containing cyanides often evolve hydrogen cyanide
     if acidified.

    Reagents

    1.   Aqueous sodium hydroxide solution (100 g/l).

    2.   Aqueous ferrous sulfate solution (100 g/l, freshly prepared in
         freshly boiled and cooled water).

    3.   Aqueous hydrochloric acid (100 ml/l).

    Method

    1.   Dilute 1 ml of sample with 2 ml of sodium hydroxide solution.

    2.   Add 2 ml of ferrous sulfate solution.

    3.   Add sufficient hydrochloric acid to dissolve the ferrous
         hydroxide precipitate.

 

   Result

         A blue colour indicates the presence of cyanide. There are no
    common sources of interference.

    Sensitivity

         Cyanide, 10 mg/l.

    Quantitative assays

         Applicable to heparinized whole blood (0.1-1.0 ml), which can be
    stored at 4C for 1-2 days if the analysis is delayed for any reason.
    (Cyanide in blood is less stable if stored at room temperature or at
    -20C.)

    1.  p-Nitrobenzaldehyde/o-dinitrobenzene method

    Reagents

    1.   Aqueous sodium hydroxide (0.5 mol/l).

    2.   Aqueous sulfuric acid (3.6 mol/l).

    3.    p-Nitrobenzaldehyde (0.05 mol/l) in 2-methoxyethanol.

    4.    o-Dinitrobenzene (0.05 mol/l) in 2-methoxyethanol.

    Standard

         Aqueous potassium cyanide (10 mg/l, i.e., cyanide ion
    concentration, 4 mg/l).

    Method

    1.   Take three microdiffusion cells (see section 4.3.3) and add to
         each of the centre wells:

         (a) 0.5 ml of  p-nitrobenzaldehyde solution;

         (b) 0.5 ml of  o-dinitrobenzene solution;

         (c) 0.1 ml of sodium hydroxide solution.

    2.   To the outer wells add 0.1 ml of:

         - purified water (cell 1);

         - potassium cyanide solution (cell 2);

         - test blood specimen (cell 3).

    3.   To each outer well add 0.5 ml of purified water and, on the
         opposite side of the outer well, 1.0 ml of dilute sulfuric acid.

    4.   Seal each well using silicone grease, and carefully mix the
         components of the outer wells.

    5.   Incubate at room temperature for 20 minutes and then add 1 ml of
         aqueous methanol (1:1) to the centre wells.

    6.   Transfer the contents of the centre wells to 5.0-ml volumetric
         flasks and make up to volume with aqueous methanol (1:1).

    Results

         The red coloration obtained with cyanide-containing solutions is
    stable for about 15 minutes. Measure the absorbance of the solutions
    from cells 2 and 3 at 560 nm against the purified water blank (cell 1;
    see section 4.5.2). Assess the cyanide ion concentration in the sample
    by comparison with the reading obtained from the standard.

    Sensitivity

         Cyanide, 0.5 mg/l.

    2.  Pyridine/barbituric acid method

    Reagents

    1.   Aqueous sodium hydroxide (0.1 mol/l).

    2.   Aqueous chloramine T solution (2.5 g/l). (N.B. Solid chloramine
         T is not stable, and fresh supplies should be obtained
         frequently.)

    3.   Aqueous sodium hydrogen orthophosphate (1 mol/l).

    4.   Pyridine-barbituric acid reagent. Stir 6 g of barbituric acid
         ( not diethyl barbituric acid, see section 6.9) into 6 ml of
         concentrated hydrochloric acid (relative density 1.18), and
         dilute with 30 ml of pyridine. Dilute the resulting solution to
         100 ml with purified water. This solution must be freshly
         prepared.

    5.   Aqueous sulfuric acid (1 mol/l).

    Standards

    1.   Cyanide stock solution. Dissolve 50 mg of potassium cyanide in
         100 ml of aqueous sodium hydroxide (0.1 mol/l); cyanide ion
         concentration, 200 mg/l.  Take care when using concentrated
          cyanide solutions.

    2.   Cyanide calibration solution. Dilute (1:99) the standard cyanide
         ion solution (200 mg/l) in aqueous sodium hydroxide (0.1 mol/l);
         final cyanide ion concentration 2 mg/l.

    Method

    1.   Label seven microdiffusion cells (a) to (g) and add the reagents
         shown in Table 25 to the outer wells, reagent 5 (dilute sulfuric
         acid) being placed at the opposite side of the well to the
         others.

    2.   Add 2 ml of sodium hydroxide solution to each inner well, seal
         the cells with silicone grease, carefully mix the contents of the
         outer wells and incubate at room temperature for 4 hours.

    3.   Pipette 1.0 ml of the sodium hydroxide solution from each of the
         inner wells into prelabelled, stoppered test-tubes.

    4.   Add the following reagents sequentially and shake to mix:

         (a) 2 ml of phosphate buffer;

         (b) 1 ml of chloramine T solution;

         (c) 3 ml of pyridine-barbituric acid reagent.

    5.   Allow to stand for 10 minutes at room temperature.

    Table 25.  Cyanide measurement by microdiffusion: outer well
               reagent additions
                                                                        

    Cell                      Cyanide       Water    Sulfuric   Blood
                              calibration   (ml)     acid       (ml)
                              solution               (ml)
                              (ml)
                                                                        

    (a) Reagent blank         --            2.0      0.5        --
    (b) Test specimen         --            1.0      0.5        1.0
    (c) Test specimen         --            1.0      0.5        1.0
    (d) Cyanide, 0.2 mg/l     0.1           1.9      0.5        --
    (e) Cyanide, 1.0 mg/l     0.5           1.5      0.5        --
    (f) Cyanide, 2.0 mg/l     1.0           1.0      0.5        --
    (g) Cyanide, 4.0 mg/l     2.0           --       0.5        --
                                                                        
    Results

         The presence of cyanide is indicated by a red/blue colour.
    Measure the absorbance at 587 nm of each solution against the purified
    water blank (section 4.5.2), diluting if necessary to bring the test
    reading on to the scale. Construct a calibration graph using the
    results obtained from the standard cyanide solutions and calculate the
    cyanide concentration in the sample.

    Sensitivity

         Cyanide, 0.2 mg/l.

    Clinical interpretation

         Acute cyanide poisoning is characterized by ataxia, headache,
    anxiety, dyspnoea, confusion, coma, collapse, metabolic acidosis,
    pulmonary oedema and respiratory arrest. Cyanosis may not be present.
    Supportive treatment includes the administration of oxygen. A number
    of antidotes have been used, including dicobalt edetate,
    hydroxocobalamin, sodium nitrite and sodium thiosulfate.

         In serious poisoning with cyanide salts or hydrocyanic
    acid, blood cyanide ion concentrations are usually of the order of
    2-10 mg/l. The qualitative test described above has insufficient
    sensitivity to detect these concentrations and should only be used for
    stomach contents and scene residues. Quantitative cyanide measurements
    have little immediate relevance to the treatment of acute poisoning
    since, to have any hope of success, therapy must be commenced as soon
    as possible.

         Cyanide may also be present in the blood of fire victims owing to
    inhalation of hydrogen cyanide from the partial combustion of wool,
    silk and synthetic polymers such as polyurethanes and
    polyacrylonitriles. In such cases, blood cyanide concentrations may
    range from 0.2 to 1.0 mg/l. Carbon monoxide is usually also present.
    Blood cyanide concentrations in heavy cigarette smokers may be as high
    as 0.3 mg/l.

    6.37  Dapsone

    Bis(4-aminophenyl)sulfone; C12H12N2O2S; relative molecular mass,
    248

    CHEMICAL STRUCTURE 19

         Dapsone is a structural analogue of the sulfonamide
    antibacterials and is used in the treatment of leprosy and dermatitis
    herpetiformis. Dapsone is metabolized to monoacetyldapsone, which also
    occurs in plasma, and to a variety of other products which are largely
    excreted in urine. Enterohepatic recirculation also occurs. In an
    adult, death may ensue 4-6 days after the ingestion of 1.5-5 g of
    dapsone.

         The qualitative test described below can be used to give an
    estimate of the plasma dapsone concentration, if used with the
    appropriate standard solutions.

    Qualitative test

         Applicable to plasma or serum (0.5 ml).

    Reagents

    1.   Aqueous sodium hydroxide (1 mol/l).

    2.   Chloroform:ethanol:glacial acetic acid (90:10:5).

    3.   Silica gel thin-layer chromatography plate (10  20 cm, 20 m
         average particle size, see section 4.4.1).

    Standards

         Solutions containing dapsone concentrations of 5, 10, 20 and
    50 mg/l in blank plasma, prepared by dilution from a methanolic stock
    solution (dapsone 1.00 g/l).

    Method

    1.   Add 0.5 ml of sample or standard to 0.2 ml of sodium hydroxide
         solution and 6 ml of chloroform in a test-tube with a ground-
         glass stopper.

    2.   Stopper the tube, vortex-mix for 30 seconds and centrifuge for
         5 minutes.

    3.   Discard the upper, aqueous layer, transfer 5 ml of the chloroform
         extract to a second tube and evaporate to dryness under a stream
         of compressed air or nitrogen.

    Thin-layer chromatography

    1.   Reconstitute the extracts in 50 l of methanol and spot on the
         plate. Spot extracts of the standard dapsone solutions on
         adjacent columns on the plate.

    2.   Develop the chromatogram (10-cm run) with chloroform:ethanol:
         glacial acetic acid (saturated tank, section 4.4.3).

    3.   Remove the plate, allow to dry and inspect under ultraviolet
         light (254 nm).

    Results

         Estimate the dapsone concentration in the sample by comparison
    with the results from the standard solutions, hRf values: dapsone,
    57; monoacetyldapsone, 40.

    Sensitivity

         Dapsone, 2 mg/l.

    Clinical interpretation

         Ingestion of dapsone may cause anorexia, nausea, vomiting,
    abdominal pain, headache, tinnitus and blurred vision, with dizziness,
    agitation, coma and convulsions in severe cases. Haemolytic anaemia,
    methaemoglobinaemia, haematuria, jaundice and acute renal failure are
    further complications.

         Plasma dapsone concentrations of 10 mg/l or more may be
    associated with toxicity. Repeat-dose oral activated charcoal is
    probably the most effective method of treatment, but methylene blue
    and exchange transfusion may be needed to treat methaemoglobinaemia
    and haemolytic anaemia, respectively.

    6.38  Dextropropoxyphene

    (+)-Propoxyphene; (+)-(1S,2R)-1-benzyl-3-dimethylamino-2-methyl-1-
    phenylpropyl propionate; C22H29NO2; relative molecular mass, 340

    CHEMICAL STRUCTURE 20

         Dextropropoxyphene is a narcotic analgesic structurally
    related to methadone, and is often formulated together with
    paracetamol. Dextropropoxyphene is extensively metabolized to
     N-desmethyldextropropoxyphene (nordextropropoxyphene), which is the
    principal urinary metabolite.

         There is no simple qualitative test for dextropropoxyphene, but
    this compound and its metabolites can be detected and identified by
    thin-layer chromatography of a basic solvent extract of urine (see
    section 5.2.3). In addition, paracetamol can be detected in urine
    using the  o-cresol/ammonia test (see section 6.83).

    Clinical interpretation

         Acute overdosage with dextropropoxyphene gives rise to pin-point
    pupils, hypotension, hypothermia, coma, pulmonary oedema, convulsions
    and cardiac arrhythmias. Death may ensue rapidly from profound
    respiratory depression, especially if ethanol is also present.
    Naloxone rapidly reverses the central toxic effects of
    dextropropoxyphene (see section 2.2.2).

    6.39  Dichloralphenazone

    A stoichiometric complex of chloral hydrate and phenazone,
    C15H18Cl6N2O5; relative molecular mass, 519

    CHEMICAL STRUCTURE 21

         Dichloralphenazone is a mild hypnotic and acts as a mixture of
    each of its components, chloral hydrate and phenazone,  in 
     vivo. Dichloralphenazone ingestion can therefore be detected
    in urine using the Fujiwara test for the chloral metabolite
    trichloroacetic acid. Phenazone does not possess hypnotic activity and
    can be detected by thin-layer chromatography of a basic/neutral
    extract of urine (see section 5.2.3), using acidified iodoplatinate
    reagent only.

    Qualitative test

         Applicable to urine. Fujiwara test - see carbon tetrachloride
    monograph (section 6.23).

    Results

         An intense red/purple colour in the upper, pyridine layer
    indicates the presence of trichloro compounds. The blank analysis
    excludes contamination with chloroform from the laboratory atmosphere.

         This test is very sensitive and will detect ingestion of a
    therapeutic dose of dichloralphenazone or chloral hydrate 12-24
    hours later. However, other compounds, notably the solvent
    trichloroethylene, also give rise to trichloroacetic acid  in 
     vivo, so that caution must be exercised in reporting results.

    Sensitivity

         Trichloroacetate, 1 mg/l.

    Clinical interpretation

         Acute poisoning with dichloralphenazone can cause vomiting,
    excitement, ataxia, confusion, drowsiness, stupor, hypotension, coma,
    cardiac arrhythmias, respiratory depression and pulmonary oedema.
    Treatment is normally symptomatic and supportive.

    6.40  Dichloromethane

    Methylene chloride; CH2Cl2; relative molecular mass, 85

         Dichloromethane is widely used in paint strippers, sometimes
    together with toluene, and as a laboratory and industrial solvent. Its
    acute toxicity is largely due to direct depressant effects on the
    central nervous system. Dichloromethane is partially metabolized to
    carbon monoxide, which may contribute to chronic toxicity.

         There is no simple test for dichloromethane in biological
    samples. However, measurement of carboxyhaemoglobin saturation is
    important in the assessment of chronic exposure to dichloromethane.
    Urine samples from patients exposed to dichloromethane do not give a
    positive result with the Fujiwara test.

    Clinical interpretation

         Exposure to dichloromethane may cause dizziness, numbness,
    irritability, fatigue, nausea, hypoventilation, pulmonary oedema and
    respiratory arrest. Recovery is normally rapid once the patient is
    removed from the contaminated atmosphere. Supplemental oxygen may be
    indicated, especially if features of carbon monoxide poisoning are
    present.

    6.41  Digoxin and digitoxin

         Digoxin (C41H64O14; relative molecular mass, 781) and
    digitoxin (C41H64O13; relative molecular mass, 765) are
    cardioactive glycosides obtained from the leaves of certain species
    of  Digitalis (e.g. foxglove). Digoxin is widely used as an
    antiarrhythmic. Cardiac glycosides are also used as euthanasia agents
    in veterinary practice in certain countries. These compounds are very
    potent and there is no simple method for detecting them in blood or
    urine.

    Qualitative test

         Applicable to stomach contents and scene residues.

    Reagents

    1.   Silica gel thin-layer chromatography plate (10  20 cm, 20 m
         average particle size; see section 4.4.1).

    2.   Toluene:ethanol (7:3).

    3.   Chloramine T reagent. Mix 10 ml of aqueous chloramine T (30 g/l)
         and 40 ml of methanol containing 250 g/l trichloroacetic acid.

    4.   Aqueous perchloric acid (150 g/l).

    Standards

         Digoxin and digitoxin (both 100 mg/l) in chloroform.

    Method

    1.   Add 5 ml of chloroform to 1 ml of sample, vortex-mix for 30
         seconds and centrifuge for 5 minutes.

    2.   Discard the upper, aqueous layer and filter the chloroform
         extract through phase-separating filter-paper into a clean tube.

    3.   Evaporate the extract to dryness under a stream of compressed air
         or nitrogen and reconstitute in 50 l of chloroform.

    Thin-layer chromatography

    1.   Divide the plate into two halves and spot 20 l of the
         reconstituted extract and 10 l of the standard solutions on to
         columns on both halves of the plate.

    2.   Develop the chromatogram (10-cm run) using toluene:ethanol
         (saturated tank, see section 4.4.3).

    3.   Allow the plate to dry, and spray one half of the plate with
         chloramine T reagent and the other half with perchloric acid
         solution.

    4.   Heat the plate in an oven at 100C for 10 minutes.

    Results

         hRf values and colour reactions with the spray reagents are
    listed in Table 26.

    Table 26.  Thin-layer chromatography of digoxin and digitoxin:
               hRf values and colour reactions
                                                                        

    Compound    hRf   Chloramine T reagent      Perchloric acid (150 g/l)
                                                                         

                      Visible     Ultraviolet   Visible       Ultraviolet
                                  (366 nm)                    (366 nm)
                                                                        

    Digoxin     62    Brown ring  Blue-green    Grey          Blue-green
    Digitoxin   72    --          --            Brown         Dark brown
                                                                        

    Sensitivity

         Digoxin or digitoxin, 10 mg/l.

    Clinical interpretation

         Digoxin and digitoxin are potent cardiotoxins and can give rise
    to fatal arrhythmias. Nausea, vomiting, diarrhoea, drowsiness and
    confusion occur in the early stages of poisoning with these compounds.

    Hyperkalaemia and tachyarrhythmias are characteristic of severe
    poisoning. Treatment is generally supportive. Antigen-binding (Fab)
    antibody fragments will reverse toxicity in digoxin poisoning (see
    section 2.2.2), but are indicated only in very severe cases.

    6.42  Dinitrophenol pesticides

         These compounds have the general structure:

    CHEMICAL STRUCTURE 22

         The dinitrophenols most commonly encountered are DNOC (2-methyl-
    4,6-dinitrophenol; 4,6,-dinitro- o-cresol; C7H6N2O5; relative
    molecular mass, 198) and dinoseb (2-sec-butyl-4,6-dinitrophenol;
    C10H12N2O5; relative molecular mass, 240). DNOC is used as an
    insecticide and as a herbicide on fruit trees, while dinoseb is used
    mostly as a herbicide. Severe dinitrophenol poisoning may follow
    occupational exposure as well as ingestion. Skin is a common route of
    absorption and intense yellow staining may be diagnostic.

         Both DNOC and dinoseb can be measured easily in whole blood since
    they show strong absorbance at 430 nm and the concentrations
    associated with toxicity are relatively high.

    Quantitative assay

    Applicable to whole blood (1 ml).

    Reagents

    1.   Concentrated hydrochloric acid (relative density 1.18).

    2.   Aqueous sodium chloride (270 g/l) containing sodium carbonate
         (30 g/l).

    Standards

         Solutions containing dinitrophenol concentrations of 10, 20 and
    50 mg/l in whole blood.

    Method

    1.   Add 5 ml of butanone (methyl ethyl ketone) to 1 ml of sample or
         standard in a conical tube, and add 1 ml of the sodium chloride/
         sodium carbonate solution.

    2.   Vortex-mix for 30 seconds, centrifuge for 5 minutes and transfer
         2-ml portions of the extract to two clean tubes.

    3.   Add 50 l of hydrochloric acid to one tube, vortex-mix for 10
         seconds and centrifuge for 5 minutes.

    4.   Measure the difference in absorbance between the solutions at
         430 nm (1-cm path-length cells).

    Results

         Construct a graph of the difference in absorbance against
    dinitrophenol concentration in the calibration solutions and calculate
    the dinitrophenol concentration in the sample.

    Sensitivity

         Dinitrophenol, 10 mg/l

    Clinical interpretation

         Dinitrophenols uncouple oxidative phosphorylation, and fatigue,
    excessive sweating, hyperthermia and thirst may be followed by
    exhaustion and death in severe cases. Toxic effects often appear at
    blood concentrations greater than 30 mg/l while concentrations greater
    than 60 mg/l are associated with severe toxicity.

    6.43  Diphenhydramine

    2-Benzhydryloxy- N,N-dimethylethylamine; C17H21NO; relative
    molecular mass, 255

    CHEMICAL STRUCTURE 23

         Diphenhydramine is a widely used antihistamine. Less than 1% of
    the dose is excreted unchanged but  N-dealkylation, oxidative
    deamination and conjugation give rise to a number of compounds which
    are excreted in urine.

         There is no simple qualitative test for diphenhydramine and its
    metabolites, but this compound can be detected and identified by thin-
    layer chromatography of a basic solvent extract of urine, stomach
    contents or scene residues (see section 5.2.3).

    Clinical interpretation

         Overdosage with diphenhydramine and other antihistamines may
    cause drowsiness, dizziness, dry mouth, headache, nausea, tachycardia,
    fever, hallucinations and tremor. In more severe cases, this may be
    followed by coma, convulsions and death. Treatment is symptomatic and
    supportive.

    6.44  Diquat

    1,1'-Ethylene-2,2'-bipyridylium ion; C12H12N2; relative molecular
    mass, 184

    CHEMICAL STRUCTURE 24

         Diquat is a contact herbicide structurally related to paraquat,
    with which it is often formulated. Diquat is often encountered as the
    dibromide salt and death has been reported following the ingestion of
    as little as 2 g of diquat. Diquat and paraquat give highly coloured
    products with sodium dithionite, and this reaction forms the basis of
    the test described.

    Qualitative test

         Applicable to urine, stomach contents and scene residues.

    Reagents

    1.   Sodium dithionite (solid, stored in a desiccator).

    2.   Aqueous ammonium hydroxide (2 mol/l).

    3.   Blank urine.

    4.   Urine specimen containing diquat ion (10 mg/l).

    Method

    1.   Add 0.5 ml of ammonium hydroxide solution to the test solution
         and to the blank and standard urines (1-ml volumes) in separate
         test-tubes.

    2.   Add about 20 mg of sodium dithionite to each tube and mix.

    3.   If a colour forms in the test solution, agitate in air for
         several minutes.

    Results

         A yellow-green colour indicates diquat. Paraquat gives a
    blue/blue-black colour. This test cannot detect diquat in the presence
    of paraquat.

         If the colour fades on continued agitation in air, diquat/
    paraquat is confirmed - the original colour can be restored by adding
    more sodium dithionite.

    Sensitivity

         Diquat, 5 mg/l.

    Clinical interpretation

         Ingestion of diquat may cause irritation of the mouth and throat,
    epigastric pain, vomiting, diarrhoea, intestinal paralysis, malaise,
    excitement, convulsions, coma and hepatorenal failure. Unlike
    paraquat, diquat does not cause progressive pulmonary fibrosis.
    Treatment is largely symptomatic and supportive.

    6.45  Ephedrine

    (1 R,2 S)-2-Methylamino-1-phenylpropan-1-ol hemihydrate;
    C10H15NO(H2O)1/2; relative molecular mass, 174

    CHEMICAL STRUCTURE 25

         Ephedrine is a sympathomimetic agent. It is metabolized by
     N-demethylation to norephedrine (phenylpropanolamine), and by
    oxidative deamination and conjugation. Ephedrine is itself a
    metabolite of methylephedrine. The estimated minimum lethal dose of
    ephedrine in an adult is 4 g, but fatalities are rare.

         There is no simple qualitative test for ephedrine, but this
    compound and its metabolites can be detected and identified by thin-
    layer chromatography of a basic solvent extract of urine (see section
    5.2.3).

    Clinical interpretation

         Ephedrine overdosage may cause nausea, vomiting, headache,
    thirst, irritability, fever, tachycardia, sweating, dilated pupils,
    convulsions, coma and respiratory depression. Treatment is symptomatic
    and supportive.

    6.46  Ethanol

    Ethyl alcohol; alcohol; C2H5OH; relative molecular mass, 46

         Acute poisoning with ethanol is very frequently encountered in
    hospital admissions and is usually the result of ingestion of
    alcoholic drinks. Poisoning with industrial alcohol (methylated
    spirit) containing various denaturants, notably methanol, also occurs.

         The qualitative test described below will detect volatile
    reducing agents, of which ethanol is the most common. The quantitative
    assay is based on the oxidation of ethanol to acetaldehyde by alcohol
    dehydrogenase (ADH) in the presence of nicotinamide adenine
    dinucleotide (NAD), and is applicable to whole blood; if plasma or
    serum is used, the protein precipitation step with perchloric acid can
    be omitted. A number of manufacturers supply ethanol assay kits based
    on this reaction; if available, such kits often prove more economical
    than separate purchase of the reagents.

    Qualitative test

         Applicable to urine, stomach contents and scene residues.

    Reagent

         Potassium dichromate (25 g/l) in aqueous sulfuric acid
    (500 ml/l).

    Method

    1.   Apply 50 l of potassium dichromate solution to a strip of glass-
         fibre filter-paper and insert the paper in the neck of a test-
         tube containing 1 ml of sample.

    2.   Lightly stopper the tube and place in a boiling water-bath for
         2 minutes.

    Results

         A change in colour from orange to green indicates the presence of
    volatile reducing agents such as ethanol (see plate 7); metaldehyde,
    methanol and paraldehyde also react.

    Sensitivity

         Ethanol, 0.5 g/l.

    Quantitative assay

         Applicable to whole blood, plasma, or serum (0.5 ml).

    Reagents

    1.   Semicarbazide reagent. Dissolve 10 g of tetrasodium pyrophosphate
         decahydrate, 2.5 g of semicarbazide hydrochloride and 0.5 g of
         glycine in 250 ml of purified water. Add 10 ml of aqueous sodium
         hydroxide (2 mol/l) and dilute to 300 ml.

    2.   Aqueous nicotinamide adenine dinucleotide (NAD; also known as
         diphosphopyridine nucleotide, DPN) (13 g/l). This solution is
         stable for 2-3 months at 4C, but can be decomposed by vigorous
         agitation.

    3.   Alcohol dehydrogenase (ADH) suspension. Mix 45.5 g of ammonium
         sulfate and 3 g of tetrasodium pyrophosphate decahydrate in
         100 ml of purified water adjusted to pH 7.3 (using a pH meter)
         with either aqueous hydrochloric acid or sodium hydroxide (both
         1 mol/l) and containing 2.5 g of suspended crystalline yeast ADH;
         this solution is stable for 2-3 months at 4C.

    4.   Aqueous perchloric acid (2.9 ml of perchloric acid (700 ml/l) in
         100 ml of purified water).

    Standards

         Solutions containing ethanol concentrations of 0.5, 1.0, 2.0 and
    4.0 g/l prepared in heparinized whole blood to which 10 g/l sodium
    fluoride has been added. These solutions are stable for up to 1 month
    if stored at 4C in well-sealed containers.

    Method

    1.   Add 0.5 ml of blood to 2 ml of perchloric acid solution in a
         test-tube.

    2.   Vortex-mix for 30 seconds and then centrifuge for 5 minutes.

    3.   Add 0.1 ml of the supernatant (or 0.2 ml of an aqueous dilution
         (1:9) of plasma/serum) to a 10-ml tube containing 4.5 ml of
         semicarbazide reagent and vortex-mix for 10 seconds.

    4.   Add 0.1 ml of NAD solution and 0.02 ml of ADH suspension and mix
         gently so as not to cause foaming.

    5.   Allow to stand for 70 minutes at 20-25C and measure the
         absorbance at 340 nm against a reagent blank (see section 4.5.3).

    Results

         Construct a calibration graph of absorbance against blood ethanol
    concentration by analysis of the standard ethanol solutions and
    calculate the concentration of ethanol in the sample.

         If the specimen contains an ethanol concentration of more than
    4.0 g/l, the analysis should be repeated using a dilution (1:1 or 1:3)
    of the sample in blank plasma. Methanol does not interfere, but
    propan-2-ol and some higher alcohols will reduce NAD under the
    conditions used in this assay.

         In all cases where the analysis may be delayed, it is important
    to add 10 g/l sodium fluoride to the specimen to inhibit microbial
    metabolism (see section 5.1.6).

    Sensitivity

         Ethanol, 0.5 g/l.

    Clinical interpretation

         Ethanol is rapidly absorbed from the small intestine and can
    cause disinhibition, blurred vision, drowsiness, incoordination and
    confusion, with nausea, vomiting and coma in severe cases.
    Hypoglycaemia and convulsions may also occur, especially in young
    children. Treatment of acute poisoning is normally symptomatic and
    supportive, although dialysis may be considered in severe cases (see
    section 2.2.3).

         A simple guide to the interpretation of blood ethanol results is
    given in Table 27. However, remember that (1) ethanol potentiates the
    depressant effects on the central nervous system of many other drugs,
    and (2) chronic alcoholics may show few features of intoxication even
    with blood ethanol concentrations of 4 g/l or more. Blood ethanol
    measurements are therefore rarely helpful in the management of acute
    poisoning with this compound.

    Table 27.  Interpretation of blood ethanol concentrations
                                                                        

    Blood ethanol   Clinical features (in non-habitual users of alcohol)
    (g/l)
                                                                        

    0.5             Flushed face, euphoria; usually little apparent
                    clinical effect in adults. May be associated
                    with hypoglycaemia in young children
    1.0             Incoordination, speech defects
    1.5             Marked incoordination, staggering gait,
                    dilated pupils, nystagmus
    3.0             Gross incoordination, stupor, vomiting
    5.0             Coma
                                                                        

         Ethanol can be given to treat poisoning with ethylene glycol and
    methanol (see section 2.2.2), since it inhibits the production of
    toxic metabolites. Monitoring of the plasma ethanol concentrations
    attained is often useful in such cases, as noted in the appropriate
    monographs (sections 6.48 and 6.70).

    6.47  Ethchlorvynol

    1-Chloro-3-ethylpent-1-en-4-yn-3-ol; C7H9ClO; relative molecular
    mass, 145

    CHEMICAL STRUCTURE 26

         Ethchlorvynol is a nonbarbiturate hypnotic with a pungent smell.
    The test described is based on the reaction between ethchlorvynol and
    diphenylamine in the presence of concentrated sulfuric acid.

    Qualitative test

         Applicable to urine, stomach contents and scene residues.

    Reagents

    1.   Diphenylamine sulfate (solid).

    2.   Concentrated sulfuric acid (relative density 1.83).

    Method

    1.   Add about 20 mg of diphenylamine sulfate crystals to the surface
         of 2 ml of test solution in a glass test-tube.

    2.    Slowly add 1 ml of sulfuric acid down the side of the tube.

    Results

         A bright red colour on the surface of the crystals indicates the
    presence of ethchlorvynol. This test is specific and will detect a
    therapeutic dosage of ethchlorvynol if performed on urine.

    Sensitivity

         Ethchlorvynol, 1 mg/l.

    Clinical interpretation

         Ingestion of ethchlorvynol may cause fatigue, headache,
    confusion, nausea, vomiting, coma and respiratory depression.
    Treatment is symptomatic and supportive.

    6.48  Ethylene glycol

    Ethane-1,2-diol; glycol; CH2OH.CH2OH; relative molecular mass, 62

         Ethylene glycol is used mainly in vehicle radiator antifreeze as
    a concentrated 200-500 ml/l aqueous solution, sometimes together with
    methanol. Ethylene glycol is itself relatively nontoxic, but is
    metabolized by alcohol dehydrogenase giving rise to glycolic and
    oxalic acids. Some of the later features observed in ethylene glycol
    poisoning are therefore characteristic of poisoning with oxalates. The
    potentially fatal dose of antifreeze containing ethylene glycol in an
    adult is 50-100 ml.

         There is no simple method for the detection and identification of
    ethylene glycol in biological specimens. However, a rise in plasma
    osmolality is a useful but nonspecific indicator of poisoning with
    this compound (see section 3.1.3). Oxalic acid may be excreted in
    urine as calcium oxalate, and the tests for oxalates will detect this
    compound (see section 6.82). The crystalluria produced may also be
    diagnostic (see section 5.2.1).

    Clinical interpretation

         Ingestion of ethylene glycol may give rise initially to clinical
    features similar to those of ethanol intoxication, and inebriation,
    drowsiness, nausea, and vomiting may occur. Production of glycolate
    may give rise to a marked metabolic acidosis, which may help to

    indicate the diagnosis (see section 3.1.2). Oxalic acid itself
    sequesters calcium and hypocalcaemia, muscular twitching and tetany,
    convulsions, flank pain, acute renal failure and cardiac arrest are
    later features of severe ethylene glycol poisoning.

         Plasma concentrations of ethylene glycol of 0.5 g/l or more are
    normally associated with serious poisoning, although the time of
    ingestion is important in interpreting results. Ethanol prevents
    metabolism of ethylene glycol by competitive inhibition of alcohol
    dehydrogenase. Treatment of ethylene glycol poisoning consists of
    correction of any metabolic acidosis and of hypocalcaemia, ethanol
    administration and peritoneal dialysis or haemodialysis to treat renal
    failure and remove unchanged ethylene glycol. Plasma ethanol
    concentrations of about 1 g/l should be attained and monitored to
    ensure they are maintained during treatment since dialysis removes
    this compound as well as ethylene glycol.

    6.49  Fluoride

         Hydrogen fluoride (hydrofluoric acid, HF) and inorganic fluoride
    salts are widely used in industry and in the fluoridation of water
    supplies. Sodium fluoride (NaF), sodium fluorosilicate (Na2SiF6),
    and cryolite (Na3AlF6) are employed as insecticides and
    rodenticides. Fluorides are also used as preservatives, and in
    dentifrices and toothpastes. The estimated lethal dose of sodium
    fluoride in an adult is 1-4 g. Young children may ingest up to 0.5 g
    of fluoride by swallowing fluoride toothpaste.

         The simple tests for fluorine-containing compounds given below
    will also detect fluoroacetates such as fluoroacetamide and sodium
    fluoroacetate. Fluoride ion can be measured reliably in blood and
    urine by use of a fluoride-selective electrode. Alternatively, the
    microdiffusion technique (see section 4.3.3) described below can be
    used. This relies on the liberation of HF from the sample;
    polypropylene vessels must be used, since HF attacks glass.

    Qualitative test

         Applicable to urine, stomach contents and scene residues.

    Reagents

    1.   Aqueous sodium chloride solution (50 g/l).

    2.   Concentrated sulfuric acid (relative density 1.83).

    3.   Solid calcium hydroxide.

    4.   Powdered silica (silicon dioxide, SiO2).

    Method

    1.   Place 5 ml of sample in a porcelain crucible (10-ml volume), add
         100 mg of calcium hydroxide and evaporate gently to dryness over
         a microburner.

    2.   To destroy organic material, heat strongly until a white ash
         remains.

    3.   Add 200 mg of powdered silica and mix with the residue, stirring
         and scraping the sides of the crucible.

    4.   Spot 100 l of sodium chloride solution on to a glass microscope
         slide. Add 1 ml of concentrated sulfuric acid to the crucible and
         quickly cover with the slide, inverted so that the sodium
         chloride solution is suspended over the crucible.

    5.   Place a small beaker containing ice on the slide and heat the
         crucible gently over the microburner for 5 minutes.

    6.   Remove the slide and examine the sodium chloride solution under a
         low-power microscope.

    Results

         Volatile silicon tetrafluoride dissolves in the suspended drop to
    form sodium silicon tetrafluoride. As the water evaporates from the
    slide this forms small hexagonal crystals, sometimes with a pinkish
    tinge, which appear at the edge of the drop and before any larger,
    cubic sodium chloride crystals. This is a general test for fluorine-
    containing compounds, but is relatively insensitive.

    Sensitivity

         Fluoride, 100 mg/l.

    Confirmatory test

         Applicable to urine, stomach contents and scene residues.

    Reagents

    1.   Concentrated sulfuric acid (relative density 1.83).

    2.   Solid calcium hydroxide.

    3.   Paraffin wax.

    Method

    1.   Place 5 ml of sample in a 10-ml porcelain crucible, add 100 mg of
         calcium hydroxide and evaporate gently to dryness over a micro-
         burner.

    2.   To destroy organic material, heat strongly until a white ash
         remains.

    3.   Smear paraffin wax on to a glass microscope slide and scratch out
         a symbol (X) to expose part of the glass.

    4.   Add 1 ml of concentrated sulfuric acid to the crucible and
         quickly cover with the slide, inverted so that the exposed glass
         is over the crucible.

    5.   Remove the slide after 20 minutes and clean off the remaining wax
         with a solvent such as toluene.

    Results

         If fluorinated compounds are present, hydrogen fluoride is
    generated, which etches the exposed glass. This is a general test for
    fluorine-containing compounds, but is relatively insensitive.

    Sensitivity

         Fluoride, 100 mg/l.

    Quantitative assay

         Applicable to whole blood, plasma or serum.

    Reagents

    1.   Aqueous sulfuric acid (800 ml/l) containing 2.5 g/l tergitol.

    2.   Aqueous cerous nitrate solution (432 mg/l).

    3.   Alizarin reagent. Mix 38.5 mg of alizarin complexone, 4.2 ml of
         glacial acetic acid and 2.2 g of anhydrous sodium acetate with
         100 ml of purified water, final pH 4.3.

    4.   Polypropylene microdiffusion vessels, brink's modification,
         which has a sealing well, in addition to the inner and outer
         wells (size 68).a

              

    a    Conway polypropylene diffusion cells, Bel-Art Products,
         Pequannock, NJ 07440, USA.

    Standards

         Aqueous solutions of sodium fluoride containing fluoride ion
    concentrations of 0.5, 1.0 and 5.0 mg/l.

    Method

    1.   To each microdiffusion cell add:

         (a)  1.5 ml of sulfuric acid solution to the sealing well;

         (b)  1.0 ml of sample or standard and 1.0 ml of sulfuric acid
              solution,  without mixing, to the outer well;

         (c)  0.25 ml of cerous nitrate solution and 0.25 ml of alizarin
              reagent to the centre well.

    2.   Seal the cells, gently mix the contents of the outer wells and
         allow to stand for 3 hours at room temperature.

    Results

         A blue colour in the centre well indicates the presence of
    fluoride. The fluoride concentration in the sample can be estimated by
    comparison with the results obtained on analysis of the standard
    fluoride solutions.

    Sensitivity

         Fluoride, 0.5 mg/l.

    Clinical interpretation

         Inhalation of hydrogen fluoride may cause coughing, choking,
    fever, dyspnoea, cyanosis and pulmonary oedema. Ingestion of
    hydrofluoric acid may cause nausea, vomiting, diarrhoea and abdominal
    pain, while skin contact may give deep and painful ulceration.
    Systemic toxic effects include weakness, tetany, convulsions,
    respiratory depression and acute hepatorenal failure. Treatment is
    symptomatic and may include intensive supportive measures.

         Ingestion of fluoride salts may give rise to a burning sensation
    in the mouth and throat, dysphagia, thirst, excessive salivation,
    vomiting and diarrhoea. In severe cases muscle cramps, weakness and
    tremor may be followed by respiratory and cardiac failure. Plasma
    fluoride concentrations are normally less than 0.2 mg/l; urine
    concentrations are usually less than 1 mg/l, but up to 4 mg/l is not
    considered harmful. Blood concentrations of 2.6 mg/l or more have been
    recorded in fatalities.

    6.50  Fluoroacetate

         Sodium fluoroacetate (CF3.COONa) and fluoroacetamide
    (CF3.CONH2) are used mainly as rodenticides. Fluoroacetic acid is
    the toxic principle of  Dichapetalum cymosum (gifblaar), a plant
    endemic to southern Africa. Fluoroacetates block the tricarboxylic
    acid cycle and are extremely toxic - the approximate lethal oral dose
    for adults is 30 mg.

         Both the qualitative tests described below rely on the generation
    and subsequent detection of volatile fluorine derivatives from
    fluoroacetates present in the sample.

    Qualitative test

         Applicable to stomach contents, and scene residues. See fluoride
    monograph (section 6.49).

    Results

         Volatile silicon tetrafluoride dissolves in the suspended drop to
    form sodium silicon tetrafluoride. As the water evaporates from the
    slide this forms small hexagonal crystals, sometimes with a pinkish
    tinge, which appear at the edge of the drop and before any larger,
    cubic sodium chloride crystals. This is a general test for fluorine-
    containing compounds, but is relatively insensitive.

    Sensitivity

         Fluoroacetate, 100 mg/l.

    Confirmatory test

         Applicable to stomach contents and scene residues.

         See fluoride monograph (section 6.49).

    Results

         If fluorinated compounds are present, hydrogen fluoride is
    generated, which etches the exposed glass. This is a general test for
    fluorine-containing compounds, but is relatively insensitive.

    Sensitivity

         Fluoroacetate, 100 mg/l.

    Clinical interpretation

         Exposure to fluoroacetates may cause nausea and apprehension,
    which may be followed by tremor, cardiac arryhthmias, convulsions and
    coma. The onset of symptoms may be delayed by 0.5-2 hours. Death
    occurs from respiratory and cardiac failure, often associated with
    pulmonary oedema. Treatment is symptomatic and supportive.

    6.51  Formaldehyde

         Formaldehyde (HCHO; relative molecular mass, 30) is a colourless,
    inflammable gas and is normally encountered as an aqueous solution
    (formalin, 340-380 ml/l), which also contains methanol as a
    stabilizer. Formalin is used as a disinfectant, an antiseptic and a
    tissue-fixing and embalming fluid. Polymerized formaldehyde
    (paraformaldehyde) is used as a fumigant, and other polymeric forms
    are used as adhesives in chipboard and plywood, and in the preparation
    of insulation materials.

         Formaldehyde is rapidly metabolized  in vivo to formate and is
    itself a metabolite of methanol. Acute formaldehyde poisoning is
    uncommon, but 30 ml of formalin may be fatal in an adult.

    Qualitative test

         Applicable to stomach contents and scene residues.

    Reagents

    1.   Concentrated sulfuric acid (relative density 1.83).

    2.   Chromotropic acid (solid).

    Method

    1.   Add about 100 mg of chromotropic acid to 0.5 ml of test solution
         and vortex-mix for 5 seconds.

    2.    Carefully add 1.5 ml of concentrated sulfuric acid.

    Results

         A purple-violet colour indicates the presence of formaldehyde.

    Sensitivity

         Formaldehyde, 20 mg/l.

    Clinical interpretation

         Formaldehyde vapour is very irritating and inhalation may cause
    conjunctivitis, coughing and laryngeal and pulmonary oedema. Ingestion
    of formaldehyde solution may give rise to abdominal pain, vomiting,
    diarrhoea, hypotension, coma, metabolic acidosis and acute renal
    failure. Treatment is normally symptomatic and supportive.

    6.52  Formic acid and formate

         Formic acid (HCOOH; relative molecular mass, 46), a colourless,
    aqueous solution, is very corrosive. Many proprietary descaling agents
    contain 500-600 ml/l formic acid. Formates such as sodium formate
    (HCOONa) are used as synthetic intermediates and in the dying,
    printing and tanning industries. Formic acid is itself a metabolite of
    methanol and formaldehyde. The minimum lethal dose of formic acid in
    an adult is thought to be about 30 ml.

         The initial test given below should be used if formic acid is
    suspected. In the confirmatory test both formic acid and formates are
    reduced to formaldehyde, which can then be detected by reaction with
    chromotropic acid.

    Qualitative test

         Applicable to stomach contents and scene residues.

    Reagents

    1.   Citric acid/acetamide reagent. Citric acid (5 g/l) and acetamide
         (100 g/l) in propan-2-ol.

    2.   Aqueous sodium acetate (300 g/l).

    3.   Acetic anhydride.

    Method

    1.   Add 0.5 ml of test solution to 1 ml of citric acid/acetamide
         reagent and then add 0.1 ml of sodium acetate solution and 3.5 ml
         of acetic anhydride.

    2.   Vortex-mix for 5 seconds and heat in a boiling water-bath for 10
         minutes.

    Results

         A red colour indicates the presence of formic acid. Formaldehyde
    and formate salts do not react in this test.

    Sensitivity

         Formic acid, 50 mg/l.

    Confirmatory test

         Applicable to stomach contents and scene residues.

    Reagents

    1.   Aqueous hydrochloric acid (2 mol/l).

    2.   Magnesium powder.

    3.   Chromotropic acid (solid).

    4.   Concentrated sulfuric acid (relative density 1.83).

    Method

    1.   Add 0.1 ml of dilute hydrochloric acid to 0.1 ml of test solution
         and vortex-mix for 5 seconds.

    2.   Slowly add about 100 mg of magnesium powder until the evolution
         of gas ceases.

    3.   Add about 100 mg of chromotropic acid and vortex-mix for 5
         seconds.

    4.    Carefully add 15 ml of concentrated sulfuric acid and heat in a
         water-bath at 60C for 10 minutes.

    Results

         A purple-violet colour indicates the presence of formates or
    formic acid. Formaldehyde reacts without prior reduction.

    Sensitivity

         Formate, 50 mg/l.

    Clinical interpretation

         Formic acid is very corrosive to tissues, and ingestion may cause
    burning and ulceration of the mouth and throat, corrosion of the
    glottis, oesophagus and stomach, metabolic acidosis, intravascular
    haemolysis, disseminated intravascular coagulation, circulatory
    collapse and renal and respiratory failure. Treatment is symptomatic
    and supportive.

    6.53  Glutethimide

    2-Ethyl-2-phenylglutarimide; C13H15NO2; relative molecular mass, 217

    CHEMICAL STRUCTURE 27

         Glutethimide is a nonbarbiturate hypnotic which is now little
    used because of the risk of toxicity. The estimated minimum lethal
    dose of glutethimide in an adult is 5 g. Glutethimide forms a number
    of metabolites in humans, many of which are excreted in urine. Less
    than 2% of a dose is excreted unchanged.

         There is no simple qualitative test for glutethimide, but this
    compound and its metabolites can be detected and identified by thin-
    layer chromatography of an acidic solvent extract of urine (see
    section 5.2.3). Glutethimide is unstable at pH 11 and above, and the
    rapid decline in absorbance at 240 nm observed when a solution
    containing this compound is treated with concentrated ammonium
    hydroxide (see section 6.9) provides a further means of
    identification.

    Clinical interpretation

         Acute ingestion of glutethimide may cause dilated and unreactive
    pupils, hypotension, severe metabolic acidosis, coma, cerebral oedema,
    papilloedema and acute respiratory failure. Treatment is generally
    symptomatic and supportive. Charcoal haemoperfusion may be indicated
    in severe cases.

    6.54  Glyceryl trinitrate

    Trinitroglycerin; nitroglycerin; propane-1,2,3-triol trinitrate;
    C3H5N3O9; relative molecular mass, 227

         Glyceryl trinitrate is used as a vasodilator in the treatment of
    angina and as an explosive in dynamite. Other organic nitro compounds
    (amyl nitrite, butyl nitrite) are also vasodilators and are commonly
    abused. The estimated minimum lethal dose of glyceryl trinitrate in an
    adult is 2 g. Glyceryl trinitrate is rapidly metabolized  in vivo to
    dinitrates and mononitrates. About 20% of a sublingual dose is
    excreted in urine in 24 hours, mainly as the mononitrate.

     Take care - glyceryl trinitrate explodes on rapid heating or impact
    and is unsafe in alcoholic solution.

    Qualitative test

         Applicable to stomach contents and scene residues.

    Reagents

    1.   Diphenylamine (10 ml/l) in concentrated sulfuric acid (relative
         density 1.83).

    2.   Silica gel thin-layer chromatography plate (5  20 cm, 20 m
         average particle size; see section 4.4.1).

    Standard

         Aqueous glyceryl trinitrate (20 mg/l).

    Method

    1.   Add 4 ml of chloroform:propan-2-ol (9:1) to 1 ml of sample or
         standard in a glass-stoppered test-tube.

    2.   Vortex-mix for 1 minute, allow to stand for 1 minute and discard
         the upper (aqueous) layer.

    3.   Filter the chloroform extract through phase-separating filter-
         paper into a clean tube and evaporate to dryness without heating
         under a stream of compressed air or nitrogen.

    Thin-layer chromatography

    1.   Reconstitute the extract in 100 l of chloroform and spot 20 l
         of the sample and standard extracts on adjacent columns of the
         plate.

    2.   Develop the chromatogram in chloroform:acetone (4:1) (10-cm run,
         saturated tanks, see section 4.4.3).

    3.   Allow to dry and spray with diphenylamine solution.

    Results

         Glyceryl trinitrate (hRf 0.71) gives a blue spot on a white
    background.

    Sensitivity

         Glyceryl trinitrate, 5 mg/l.

    Clinical interpretation

         Many of the signs and symptoms of poisoning with glyceryl
    trinitrate and other organic nitrates and nitrites are similar to
    those observed with inorganic nitrates and nitrites. Thus acute
    poisoning with organic nitrates or nitrites may cause headache,
    nausea, vomiting, diarrhoea, abdominal pain, flushing, dizziness,
    confusion, hypotension, collapse, coma and convulsions.
    Methaemoglobinaemia can be produced and this may be indicated by dark
    chocolate-coloured blood (see section 3.2.2). Blood methaemoglobin can
    be measured, but is unstable and the use of stored samples is
    unreliable. Treatment is symptomatic and supportive.

    6.55  Haloperidol

    4-[4-(4-Chlorophenyl)-4-hydroxypiperidino]-4'-fluorobutyrophenone;
    C21H23ClFNO2; relative molecular mass, 376

    CHEMICAL STRUCTURE 28

         Haloperidol is a neuroleptic used orally or parenterally to treat
    schizophrenia and a variety of other disorders. Haloperidol is slowly
    excreted in urine following oral dosage, about 40% being eliminated
    within 5 days, about 1% as unchanged drug.

         There is no simple qualitative test for haloperidol, but it can
    be detected and identified by thin-layer chromatography of a basic
    solvent extract of stomach contents or scene residues (see section
    5.2.3). Urinary concentrations are often below the limit of detection
    of this method, even after overdosage.

    Clinical interpretation

         Acute overdosage with haloperidol and other butyrophenones may
    cause drowsiness, hypotension, dystonic reactions and akathisia.
    Treatment is largely symptomatic and supportive.

    6.56  Hydroxybenzonitrile herbicides

         The hydroxybenzonitriles encountered most commonly are bromoxynil
    (3,5-dibromo-4-hydroxybenzonitrile; C7H3Br2NO; relative molecular
    mass, 277) and ioxynil (3,5-diiodo-4-hydroxybenzonitrile; C7H3I2NO;
    relative molecular mass, 371).

    CHEMICAL STRUCTURE 29

         These compounds are contact herbicides with some systemic
    activity, and are widely used on cereal crops. Both bromoxynil and
    ioxynil uncouple oxidative phosphorylation so that poisoning with
    these compounds follows a similar course to that with other uncouplers
    such as the dinitrophenol pesticides and pentachlorophenol, and may
    follow occupational exposure as well as oral ingestion.

         There are no simple tests for these compounds. However, both
    bromoxynil and ioxynil show high ultraviolet absorbance at 255 nm, and
    this forms the basis of the quantitative assay outlined below.

    Quantitative assay

         Applicable to plasma or serum (1.0 ml).

    Reagent

         Aqueous trichloroacetic acid (10 g/l).

    Standards

         Solutions containing either compound at concentrations of 20, 50,
    100, 200 and 400 mg/l in blank human plasma.

    Method

    1.   Add 1 ml of trichloroacetic acid solution to 1 ml of sample or
         standard in a 10-ml test-tube fitted with a ground-glass stopper.

    2.   Add 5 ml of methyl tertiary-butyl ether, vortex-mix for 30
         seconds and centrifuge for 5 minutes.

    3.   Remove the upper (ether) layer and filter through phase-
         separating filter-paper into a clean tube.

    4.   Measure the absorbance at 255 nm against a blank plasma extract
         (see section 4.5.2).

    Results

         Construct a calibration graph of absorbance against
    hydroxybenzonitrile concentration in the calibration standards and
    calculate the hydroxybenzonitrile concentration in the sample. Care
    must be taken to minimize loss of methyl tertiary-butyl ether by
    evaporation before the absorption of the extract is measured.

         Chlorophenoxy herbicides and other compounds that are highly
    soluble in water do not interfere. However, if a scanning
    spectrophotometer is available, comparison of the absorption spectra
    of sample and standard extracts at 220-300 nm may reveal the presence
    of other interfering compounds (see section 4.5.2).

    Sensitivity

         Bromoxynil or ioxynil, 20 mg/l.

    Clinical interpretation

         Absorption of bromoxynil and ioxynil may give rise to fatigue,
    irritability, excessive sweating, hyperthermia, tachycardia, vomiting
    and thirst, which may be followed by exhaustion and cardiorespiratory
    arrest. However, there is no characteristic staining of the skin as
    with the dinitrophenol pesticides. Treatment is largely symptomatic
    and supportive. As with the chlorophenoxy herbicides, alkalinization
    may protect against the systemic toxicity of these compounds. Plasma
    concentrations of either compound greater than 20 mg/l may be
    associated with clinical features of toxicity.

    6.57  Hypochlorites

         Hypochlorites such as sodium hypochlorite (NaOCl) and calcium
    hypochlorite (bleaching powder, chlorinated lime, Ca(OCl)2) are
    widely used in bleach and disinfectant solutions. Domestic bleach is a
    30-60 g/l aqueous solution of sodium hypochlorite, but higher
    concentrations (200 g/l) may be used, for example, to chlorinate
    swimming-pools.

         Hypochlorites are strong oxidizing agents, and the test given
    below will also detect compounds with similar properties, such as
    bromates, chlorates, iodates, nitrates, and nitrites.

    Qualitative test

         Applicable to stomach contents and scene residues.

    Reagent

         Diphenylamine (10 g/l) in concentrated sulfuric acid (relative
    density 1.83).

    Method

    1.   Filter, if necessary, 5 ml of stomach contents into a 10-ml glass
         tube.

    2.   Place 0.5 ml of filtrate or scene residue in a clean tube and
         slowly add 0.5 ml of diphenylamine solution down the side of the
         tube so that it forms a layer under the sample.

    Results

         A true positive is indicated by a strong blue colour which
    develops immediately at the junction of the two layers. A light blue
    colour will be given by most samples of stomach contents owing to the
    presence of organic material. As all strong oxidizing agents are
    rapidly reduced in biological samples, the test should be performed as
    soon as possible after receipt of the sample.

         In contrast to other strong oxidizing agents, hypochlorites tend
    to evolve noxious green chlorine gas ( take care) when treated with
    concentrated sulfuric acid, and this is a further diagnostic feature.

    Sensitivity

         Hypochlorite, 10 mg/l.

    Confirmatory tests

         Applicable to stomach contents and scene residues.

    1.  Lead acetate test

    Reagents

    1.   Glacial acetic acid.

    2.   Aqueous lead acetate solution (50 g/l).

    Method

    1.   Add acetic acid drop by drop to 1 ml of test solution to give a
         final pH of about 6 (universal indicator paper).

    2.   Add 0.5 ml of lead acetate solution and boil for 2-3 minutes.

    Results

         A brown precipitate confirms hypochlorite. Sulfides give an
    immediate brown/black precipitate with lead acetate solution.

    Sensitivity

         Hypochlorite, 10 mg/l.

    2.  Potassium iodide/starch test

    Reagents

    1.   Aqueous potassium iodide solution (100 g/l).

    2.   Glacial acetic acid.

    3.   Starch (solid).

    Method

    1.   Add 0.1 ml of test solution to 0.1 ml of acetic acid and then add
         0.1 ml of potassium iodide solution.

    2.   Mix and add about 20 mg of starch.

    Results

         A blue colour confirms hypochlorite.

    Sensitivity

         Hypochlorite, 10 mg/l.

    Clinical interpretation

         Ingestion of hypochlorites may lead to the formation of
    hypochlorous acid by reaction with gastric acid, and this in turn may
    release free chlorine which may be inhaled. Features of poisoning with
    hypochlorites therefore include nausea, vomiting, diarrhoea, abdominal
    pain, confusion, hypotension, coma and pulmonary oedema. Irritation
    and corrosion of the mucous membranes, and oesophageal and gastric
    perforation may also occur, especially with more concentrated
    formulations. Treatment is symptomatic and supportive.

    6.58  Imipramine

    3-(10,11-Dihydro-5 H-dibenz [b,f]azepin-5-yl)- N,N-dimethylpropylamine;
    C19H24N2; relative molecular mass, 280

    CHEMICAL STRUCTURE 30

         Imipramine is a widely used tricyclic antidepressant; it is
    metabolized by  N-demethylation to desipramine, which is also used as
    an antidepressant in its own right. Trimipramine and clomipramine are
    analogues of imipramine.

         The test described below (Forrest test) is based on the reaction
    of these compounds with acidified potassium dichromate solution.

    Qualitative test

         Applicable to urine, stomach contents and scene residues.

    Reagent

         Forrest reagent. Mix 25 ml of aqueous potassium dichromate
    solution (2 g/l) with 25 ml of aqueous sulfuric acid (300 ml/l), 25 ml
    of aqueous perchloric acid (200 g/kg) and 25 ml of aqueous nitric acid
    (500 ml/l).

    Method

         Add 1 ml of Forrest reagent to 0.5 ml of urine and vortex-mix for
    5 seconds.

    Results

         A yellow-green colour deepening through dark green to blue
    indicates the presence of imipramine, desipramine, trimipramine or
    clomipramine. Phenothiazines may interfere and the FPN test for these
    latter compounds should also be performed (see section 6.91).

         When applied to urine, this test will detect only acute
    overdosage. As with other tricyclic antidepressants, such as
    amitriptyline, greater sensitivity and selectivity can be obtained by
    thin-layer chromatography of a basic urine extract (see section
    5.2.3), which should always be performed if possible.

    Sensitivity

         Imipramine, 25 mg/l.

    Clinical interpretation

         Acute poisoning with tricyclic antidepressants, such as
    imipramine, may be associated with dilated pupils, hypothermia,
    cardiac arrhythmias, respiratory depression, convulsions, coma and
    cardiorespiratory arrest. Urinary retention is also a feature of
    poisoning with these compounds, and this may delay procurement of an
    appropriate specimen for analysis.

         Treatment is generally symptomatic and supportive. The use of
    antiarrhythmic agents is generally avoided, but alkalinization using
    sodium bicarbonate is sometimes employed. Quantitative measurements in
    blood are not normally required in management.

    6.59  Iodates

         Iodates such as potassium iodate (KIO3) and sodium iodate
    (NaIO3) are used as disinfectants, food additives, dietary
    supplements and chemical reagents. Iodates are strong oxidizing
    agents, and the test given below will also detect compounds with
    similar properties, such as bromates, chlorates, hypochlorites,
    nitrates and nitrites.

    Qualitative test

         Applicable to stomach contents and scene residues.

    Reagent

         Diphenylamine (10 g/l) in concentrated sulfuric acid (relative
    density 1.83).

    Method

    1.   Filter, if necessary, 5 ml of stomach contents into a 10-ml glass
         tube.

    2.   Place 0.5 ml of filtrate or scene residue in a clean tube and
          slowly add 0.5 ml of diphenylamine solution down the side of
         the tube so that it forms a layer under the sample.

    Results

         A true positive is indicated by a strong blue colour which
    develops immediately at the junction of the two layers. A light blue
    colour will be given by most samples of stomach contents owing to the
    presence of organic material. As all strong oxidizing agents are
    rapidly reduced in biological samples, the test should be performed as
    soon as possible after receipt of the sample.

    Sensitivity

         Iodate, 1 mg/l.

    Confirmatory test

         Applicable to stomach contents and scene residues.

    Reagents

    1.   Aqueous acetic acid (50 ml/l).

    2.   Aqueous starch solution (10 g/l, freshly prepared).

    3.   Aqueous potassium thiocyanate solution (50 g/l).

    Method

    1.   To 0.1 ml of starch solution add 0.3 ml of purified water and
         0.1 ml of potassium thiocyanate solution.

    2.   Mix well and add 0.1 ml of test solution acidified with 0.1 ml of
         acetic acid solution.

    Results

         A blue colour is specific for iodate. Iodate also reacts like
    chloride in the confirmatory test for bromates (see section 6.14).

    Sensitivity

         Iodate, 100 mg/l.

    Clinical interpretation

         Acute poisoning with iodates may cause nausea, vomiting,
    diarrhoea, abdominal pain, confusion, coma and convulsions.
    Methaemoglobinaemia is often produced and this may be indicated by
    dark chocolate-coloured blood (see section 3.2.2). Blood
    methaemoglobin can be measured, but is unstable and the use of stored
    samples is unreliable. Treatment is symptomatic and supportive.

    6.60  Iodine and iodide

         Iodine (I2) is one of the oldest antiseptics in medicine. It is
    used topically as a solution in ethanol (tincture) containing
    elemental iodine together with potassium iodide (KI) or sodium iodide
    (NaI), which enhances the solubility of iodine itself by forming
    polyiodide ion. Potassium iodide and sodium iodide are themselves used
    as dietary supplements and in photography, but are relatively
    innocuous in comparison with iodine.

         When iodine is applied to the skin or mucous membranes it is
    absorbed as iodide. The qualitative test given below serves to
    indicate the presence of inorganic iodides or bromides and the
    appropriate confirmatory tests must then be used.

    Qualitative test

         Applicable to urine, stomach contents and scene residues.

    Reagents

    1.   Aqueous nitric acid (2 mol/l).

    2.   Aqueous silver nitrate solution (10 g/l).

    3.   Concentrated ammonium hydroxide (relative density 0.88).

    Method

    1.   Add 0.1 ml of nitric acid to 1 ml of clear test solution, mix and
         add 0.1 ml of silver nitrate solution.

    2.   Centrifuge to isolate any significant precipitate and treat with
         0.1 ml of ammonium hydroxide solution.

    Results

         A white precipitate soluble in ammonium hydroxide indicates
    chloride, an off-white precipitate sparingly soluble in ammonium
    hydroxide indicates bromide, and a creamy-yellow, insoluble
    precipitate indicates iodide.

    Sensitivity

         Iodide, 100 mg/l.

    Confirmatory test

         Applicable to urine, stomach contents and scene residues.

    Reagents

    1.   Aqueous hydrochloric acid (2 mol/l).

    2.   Starch (solid).

    3.   Sodium nitrite solution (100 g/l, freshly prepared).

    Method

         Vortex-mix 0.1 ml of test solution, about 20 mg of starch, 0.1 ml
    of dilute hydrochloric acid and 0.1 ml of sodium nitrite solution in a
    test-tube.

    Results

         A blue colour confirms iodide.

    Sensitivity

         Iodide, 100 mg/l.

    Clinical interpretation

         Acute poisoning with iodine solutions may cause corrosion of the
    mucous membranes of the mouth, oesophagus and stomach, vomiting,
    diarrhoea and abdominal pain. In severe cases, delirium, coma,
    circulatory collapse and acute renal failure may ensue. Absorption of
    as little as 2-4 g of free iodine may cause death. Treatment is
    generally symptomatic and supportive. Starch may be administered to
    adsorb orally ingested iodine.

         Acute ingestion of iodide salts may cause angioedema, swelling of
    the larynx and cutaneous haemorrhages. However, as with bromides,
    signs of toxicity are more likely to occur with chronic poisoning, and
    include the presence of a burning sensation in the mouth and throat,
    metallic taste, sore teeth and gums, hypersalivation, headache,
    pulmonary oedema, enlargement of the parotid and submaxillary glands,
    anorexia, diarrhoea, fever and depression. Treatment is symptomatic
    and supportive.

    6.61  Iron

         Ferrous (iron II) salts are used in the treatment of iron
    deficiency anaemia and ferric (iron III) salts, which are more toxic,
    have been used as abortifacients. The minimum lethal dose of ferrous
    sulfate in an adult is of the order of 30 g, but 1 g may be dangerous
    in an infant. A green or blue colour in vomit or stomach contents
    suggests the presence of iron or copper salts.

         The qualitative test given below can be used to differentiate
    between ferrous and ferric iron and other metals, while the
    quantitative assay can be used to measure serum iron. It is very
    important to avoid contamination when collecting blood for the
    measurement of serum iron concentrations; vigorous discharge of the
    sample through the syringe needle can cause sufficient haemolysis to
    invalidate the assay.

    Qualitative test

         Applicable to stomach contents and scene residues.

    Reagents

    1.   Aqueous hydrochloric acid (2 mol/l).

    2.   Aqueous potassium ferricyanide solution (10 g/l).

    3.   Aqueous potassium ferrocyanide solution (10 g/l).

    Method

    1.   To 0.1 ml of sample add 0.1 ml of dilute hydrochloric acid and
         0.05 ml of potassium ferricyanide solution and vortex-mix for
         5 seconds.

    2.   To a further 0.1 ml of sample add 0.1 ml of dilute hydrochloric
         acid and 0.05 ml of potassium ferrocyanide solution, and vortex-
         mix for 5 seconds.

    3.   Leave for 5 minutes at ambient temperature and centrifuge for 5
         minutes.

    Results

         Deep blue precipitates with potassium ferricyanide (step 1) and
    potassium ferrocyanide (step 2) indicate the presence of ferrous and
    ferric iron, respectively.

    Sensitivity

         Ferrous or ferric iron, 10 mg/l.

    Quantitative assay

         Applicable to unhaemolysed serum (2 ml).

    Reagents

    1.   Aqueous sodium sulfite solution (0.1 mol/l, freshly prepared).

    2.   2,2'-Bipyridyl (1 g/l) in aqueous acetic acid (30 ml/l).

    3.   Aqueous hydrochloric acid (0.005 mol/l).

    Standards

         Prepare aqueous solutions containing ferrous ion concentrations
    of 1.0, 2.0, 5.0 and 10.0 mg/l by dilution of ferrous ammonium sulfate
    solution (1.00 g of ferrous iron per litre) with dilute hydrochloric
    acid.

    Method

    1.   Mix 2 ml of sample, 2 ml of sodium sulfite solution and 2 ml of
         2,2'-bipyridyl solution in a 10-ml centrifuge tube with a ground-
         glass neck.

    2.   Heat in a boiling water-bath for 5 minutes, cool and add 1 ml of
         chloroform.

    3.   Stopper, rotary-mix for 5 minutes and then centrifuge for 5
         minutes.

    4.   If an emulsion forms, vortex-mix for 30 seconds and repeat the
         centrifugation.

    5.   Remove the chloroform extract, filter through phase-separating
         filter-paper and measure the absorbance of the extract at 520 nm
         against a reagent blank (see section 4.5.2).

    Results

         Construct a calibration graph of absorbance against ferrous iron
    concentration in the calibration standards and calculate the iron
    concentration in the sample.

         This method should not be used if chelating agents such as
    deferoxamine have been given before the specimen was obtained, since
    the result may not be reliable.

    Sensitivity

         Iron, 0.5 mg/l.

    Clinical interpretation

         Acute poisoning with iron salts is extremely dangerous,
    especially in young children. The absorbed iron may rapidly exceed the
    binding capacity of transferrin so that free iron accumulates in the
    blood. Rapid necrosis of the gastrointestinal mucosa may occur with
    haemorrhage and loss of electrolytes and fluid. Treatment may include
    chelation therapy with deferoxamine, given intravenously or orally
    (see Table 6).

         Normal serum iron concentrations are less than 1.8 mg/l
    (34 mol/l). Serious toxicity may occur at concentrations above 5 mg/l
    (90 mol/l) in children or above 8 mg/l (145 mol/l) in adults. The
    serum iron concentration should be measured before and during
    chelation therapy, both to confirm the need for such treatment and to
    monitor efficacy.

    6.62  Isoniazid

    Isonicotinic acid hydrazide; INAH; INH; isonicotinohydrazide;
    C6H7N3O; relative molecular mass, 137

    CHEMICAL STRUCTURE 31

         Isoniazid is used in the treatment of tuberculosis. The principal
    metabolic reaction is acetylation, but other reactions include
    hydrolysis, conjugation with glycine and  N-methylation. Up to 70% of
    a dose is excreted in urine, largely as metabolites. Serious toxicity
    may occur in adults with doses of 3 g.

         The colorimetric procedure given below can be used to measure the
    plasma isoniazid concentration if overdosage with this drug is
    suspected.

    Quantitative assay

         Applicable to plasma or serum (2 ml).

    Reagents

    1.   Aqueous metaphosphoric acid (200 g/l).

    2.   Aqueous acetic acid (2 mol/l).

    3.   Sodium nitroprusside reagent. Mix 25 ml of aqueous sodium
         nitroprusside (20 g/l) with 25 ml of aqueous sodium hydroxide
         (4 mol/l), freshly prepared.

    Standards

         Standard solutions containing isoniazid concentrations of 5, 10,
    20 and 50 mg/l in blank plasma.

    Method

    1.   Add 4 ml of purified water to 2 ml of sample and add 2 ml of
         dilute metaphosphoric acid.

    2.   Vortex-mix for 30 seconds and allow to stand for 10 minutes.

    3.   Centrifuge for 5 minutes and transfer 4 ml of the supernatant to
         a clean tube.

    4.   Add 2 ml of dilute acetic acid, 2 ml of sodium nitroprusside
         reagent and vortex-mix for 5 seconds.

    5.   Allow to stand for 2 minutes and measure the absorbance at 440 nm
         against a plasma blank (see section 4.5.2).

    Results

         Prepare a calibration graph by analysis of the standard isoniazid
    solutions and calculate the isoniazid concentration in the sample.
    Specimens containing isoniazid at concentrations greater than 50 mg/l
    should be diluted with blank plasma and re-analysed.

         Related drugs such as iproniazid and pyrazinamide interfere in
    this test. 4-Aminosalicylate also interferes, but only if present at a
    high concentration.

    Sensitivity

         Isoniazid, 5 mg/l.

    Clinical interpretation

         Overdosage with isoniazid may cause nausea, vomiting, dilated
    pupils, hypotension, hyperglycaemia, oliguria, metabolic acidosis,
    coma, convulsions, and circulatory and respiratory failure. Treatment
    is generally symptomatic and supportive, although intravenous
    administration of pyridoxine (vitamin B6) is indicated in severe
    cases (see Table 4).

         The plasma isoniazid concentrations attained during therapy are
    normally less than 10 mg/l. Toxicity has been associated with plasma
    concentrations greater than 20 mg/l, and blood concentrations of up to
    150 mg/l have been reported in fatalities.

    6.63  Laxatives

         Laxatives are sometimes abused by patients with eating disorders
    such as bulimia or anorexia nervosa. Some commonly encountered
    laxatives are listed in Table 28. Bisacodyl, dantron and
    phenolphthalein are synthetic compounds, while rhein is a common
    constituent of many laxatives of vegetable origin (senna, cascara,
    frangula and rhubarb root).

        Table 28.  Some common laxatives
                                                                             

    Compound           Chemical name                               Relative
                                                                   molecular
                                                                     mass
                                                                             

    Bisacodyl          4,4'-(2-Pyridylmethylene)di(phenyl acetate)   361
    Dantron            1,8-Dihydroxyanthraquinone                    240
    Phenolphthalein    3,3-Bis(4-hydroxyphenyl)phthalide             318
    Rhein              9,10-Dihydro-4,5-dihydroxy-9,10-              284
                         dioxo-2-anthracene carboxylic acid
                                                                             
    
         The test given below, which involves thin-layer chromatography of
    a solvent extract of a hydrolysed urine specimen, is especially useful
    in differentiating between diarrhoea due to microbial infection or
    food allergy and that due to self-medication with laxatives.

    Qualitative test

         Applicable to urine.

    Reagents

    1.   Aqueous sodium hydroxide solution (6 mol/l).

    2.   Sodium acetate buffer. Adjust 300 g/l of sodium acetate dihydrate
         to pH 5 with glacial acetic acid.

    3.   Ketodase solution (-glucuronidase 5000 units/ml).

    4.   Chloroform:propan-2-ol (9:1)

    5.    m-Xylene:isopropylacetone:methanol (10:10:1) (XIAM).

    6.    n-Hexane:toluene:glacial acetic acid (3:1:1) (HTAA).

    7.   Silica gel thin-layer chromatography plates (20  20 cm, 20 m
         average particle size, see section 4.4.1).

    Standards

    1.   Bisacodyl. Dissolve 2 mg in 10 ml of methanol. Add 100 l of
         sodium hydroxide solution and heat on a water-bath for 30 minutes
         at 70C to prepare the monohydroxy and dihydroxy analogues.

    2.   Dantron (2 mg in 10 ml of chloroform).

    3.   Phenolphthalein (2 mg in 200 l of methanol and 10 ml of
         chloroform).

    4.   Rhein (2 mg in 10 ml of chloroform).

         These solutions are stable for at least 2 months if stored in
    stoppered tubes at 4C.

    Method

    1.   To 20 ml of urine add 1 ml of ketodase and 2 ml of sodium acetate
         buffer.

    2.   Incubate in a water-bath for 2 hours at 60C.

    3.   Cool and add 25 ml of chloroform:propan-2-ol (9:1).

    4.   Vortex-mix for 5 minutes, centrifuge for 5 minutes and remove the
         upper, aqueous layer by aspiration.

    5.   Evaporate the organic extract to dryness under a stream of
         compressed air or, preferably, nitrogen at 40C.

    Thin-layer chromatography

    1.   Reconstitute the extract in 100 l of chloroform.

    2.   Spot 10 l of the reference solutions and 3 l and 10 l of the
         reconstituted extract on to each of two plates.

    3.   Develop plate 1 in XIAM and plate 2 in HTAA (10-cm run, saturated
         tanks, see section 4.4.3).

    4.   Inspect under ultraviolet light at 366 nm and mark the outline of
         the spots with a pencil.

    5.   Spray each plate with sodium hydroxide solution.

    Results

         The thin-layer chromatography characteristics of the compounds
    studied are given in Table 29. This method will detect a single dose
    of the named laxatives up to about 36 hours after ingestion.

    Table 29.  Thin-layer chromatography of laxatives: hRf values
               and colours
                                                                        

    Compound                hRf                Ultra-      Sodium
                                               violet      hydroxide
                            XIAM     HTAA      (366 nm)
                                                                        

    Rhein                   02       19        Orange      Red
    Bisacodyl metabolite    17       --        --          Purple/grey
    Dihydroxybisacodyl      21       --        --          Red/purple
    Monohydroxybisacodyl    26       --        --          Red/purple
    Bisacodyl               30       --        --          Red/purple
    Phenolphthalein         32       05        --          Red
    Dantron                 45       27        Orange      Yellow
                                                                        

         Note that bisacodyl is only detected after hydrolysis as a
    dihydroxy compound and as a hydroxylated metabolite at a lower hRf
    value on the XIAM plate. Both of these compounds give a purple colour
    with the sodium hydroxide spray.

    Sensitivity

         Each laxative, about 0.2 mg/l.

    Clinical interpretation

         Chronic ingestion of laxatives may lead to diarrhoea,
    hypokalaemia and large bowel dilatation, while additional problems,
    such as skin disorders (with phenolphthalein), may occur in certain
    patients.

    6.64  Lead

         Lead (Pb) and lead compounds have a number of industrial uses
    ranging from paint additives to solder, batteries and building
    materials. Well known insoluble lead compounds include red lead
    (Pb3O4) and white lead (basic lead carbonate, PbCO3.Pb(OH)2). The
    most important soluble salts of lead are lead nitrate (Pb(NO3)2) and
    lead acetate (Pb(CH3COO)2). This latter compound is also known as
    sugar of lead because of its sweet taste. Organic lead compounds, such
    as tetraethyl lead, are still used as antiknock agents in petrol.

         There is no simple qualitative test for lead that can be carried
    out on biological samples. However, physical and chemical tests on
    materials suspected of containing lead may be useful. Lead compounds
    will usually sink to the bottom when sprinkled into a glass of water,
    and this may be useful in the examination of paint flakes or
    cosmetics, such as surma (an Asian preparation often containing
    antimony or lead). It should be borne in mind that finely divided lead
    compounds may float on the surface as a result of the surface tension
    of the water.

    Qualitative test

         Applicable to stomach contents and scene residues.

    Reagents

    1.   Sodium tartrate buffer, pH 2.8. Mix sodium bitartate (19 g/l) and
         tartaric acid (15 g/l) in purified water.

    2.   Aqueous sodium rhodizonate solution (10 g/l).

    Method

    1.   Add 0.1 ml of sodium tartrate buffer to 0.1 ml of test solution
         and vortex-mix for 5 seconds.

    2.   Spot 50 l of acidified solution on to phase-separating filter-
         paper and add 50 l of sodium rhodizonate solution.

    Results

         Lead salts give a purple colour in this test. However, the test
    is not specific: barium salts give a brown colour and a number of
    other metals also give coloured complexes.

    Sensitivity

         Lead, 2 mg/l.

    Clinical interpretation

         The acute ingestion of soluble lead salts may cause severe
    colicky pain with constipation or diarrhoea. Chronic lead poisoning is
    more common and additional symptoms include fatigue, anaemia and joint
    weakness and pain. Lead poisoning in young children may cause coma and
    encephalopathy. Treatment may include chelation therapy.

         In the absence of facilities for the accurate measurement of lead
    concentration in blood, a diagnosis of lead poisoning is best made
    from a careful evaluation of the history and clinical presentation.
    Nonspecific signs that may indicate a diagnosis of chronic lead
    poisoning include basophilic stippling of red cells, a blue gum line
    and wrist drop. Specialized clinical chemical tests that may also
    assist in diagnosis include red-cell zinc protoporphyrin concentration
    and urinary delta-amino-laevulinic acid excretion, but again suitable
    facilities may not be available.

    6.65  Lidocaine

    Lignocaine; 2-diethylaminoaceto-2',6'-xylidide; C14H22N2O; relative
    molecular mass, 234

    CHEMICAL STRUCTURE 32

         Lidocaine is used as a local anaesthetic and is commonly found in
    lubricant gels for use with urinary catheters. It is also used as an
    antiarrhythmic but is only effective when given intravenously, since
    it undergoes extensive first-pass metabolism. Metabolic pathways
    include  N-dealkylation, hydroxylation, amide hydrolysis and
    glucuronide formation; only some 3% of an oral dose is excreted
    unchanged in urine. The fatal oral dose of lidocaine is about 25 g in
    an adult.

         Lidocaine is often found in urine and other samples from oisoned
    patients, sometimes in very high concentrations. This usually results
    from topical use of a lubricant gel containing lidocaine. Metabolites
    may also occur in urine following topical use of lidocaine.

         There is no simple qualitative test for lidocaine, but it can be
    detected and identified by thin-layer chromatography of a basic
    solvent extract of urine, stomach contents or scene residues (see
    section 5.2.3).

    Clinical interpretation

         Acute poisoning with lidocaine may cause confusion, paraesthesia,
    hypotension, coma, convulsions and circulatory collapse. Treatment is
    symptomatic and supportive.

    6.66  Lithium

         Lithium (Li) salts have a number of industrial uses, and lithium
    carbonate (Li2CO3; relative molecular mass, 74) and lithium citrate
    (C6H5Li3O74H2O; relative molecular mass, 282) are widely used in
    the treatment of depression and mania. Lithium is excreted in urine,
    but the plasma half-life is dependent on the duration of therapy,
    among other factors. In therapy, 2 g of lithium carbonate may be given
    daily for 5-7 days, but this is normally reduced to 0.6-1.2 g/day
    thereafter. Survival has followed the acute ingestion of 22 g of
    lithium carbonate, although this is unusual.

         There is no simple method for the measurement of lithium in
    biological specimens. The test given below can be used to indicate the
    presence of lithium salts in samples which contain relatively high
    concentrations of this element.

    Qualitative test

         Applicable to scene residues.

    Reagents

    1.   Concentrated hydrochloric acid (relative density 1.18).

    2.   Platinum wire.

    Method

    1.   Dip the end of the platinum wire in the concentrated acid.

    2.   Dip the moistened end of the wire into the test material.

    3.   Place the material in the hot part of the flame of a microburner.

    Results

         A crimson red flame denotes the presence of lithium salts.
    However, calcium and strontium salts also give red flames, while high
    concentrations of sodium (yellow flame) can mask other colours.

    Sensitivity

         Lithium, 50 mg/l.

    Clinical interpretation

         Acute poisoning with lithium salts can cause nausea, vomiting,
    apathy, drowsiness, tremor, ataxia and muscular rigidity, with coma,
    convulsions and death in severe cases. Overdosage with lithium tends
    to be more serious in patients on chronic lithium therapy, since
    tissue sites are saturated and lithium accumulates in plasma.
    Treatment is symptomatic and supportive, but peritoneal dialysis or
    haemodialysis may be indicated in severe cases (see section 2.2.3).

    6.67  Meprobamate

    2-Methyl-2- iso-propylpropane-1,3-diol dicarbamate; C9H18N2O4;
    relative molecular mass, 218

    CHEMICAL STRUCTURE 33

         Meprobamate is a sedative and tranquillizer. Metabolites include
    meprobamate  N-oxide and 2-hydroxypropylmeprobamate. About 90% of a
    dose is excreted in urine, 15% as unchanged drug. The estimated
    minimum lethal dose in an adult is 12 g, but recovery has occurred
    after much larger doses.

         The qualitative test described here is based on a general
    reaction of carbamates with furfuraldehyde in the presence of hydrogen
    chloride. The confirmatory test is also applicable to urine, and is
    based on solvent extraction followed by thin-layer chromatography of
    the concentrated extract.

    Qualitative test

         Applicable to stomach contents and scene residues.

    Reagents

    1.   Aqueous hydrochloric acid (2 mol/l).

    2.   Furfuraldehyde solution (100 ml/l) in methanol, freshly prepared.

    3.   Concentrated hydrochloric acid (relative density 1.18).

    Method

    1.   Acidify 1 ml of sample with 0.5 ml of dilute hydrochloric acid,
         and extract with 4 ml of chloroform on a rotary mixer for 5
         minutes.

    2.   Centrifuge in a bench centrifuge for 5 minutes, discard the
         upper, aqueous phase and filter the chloroform extract through
         phase-separating filter-paper into a clean tube.

    3.   Evaporate the extract to dryness under a stream of compressed air
         or nitrogen at 40C.

    4.   Dissolve the residue in 0.1 ml of methanol and apply a 10-mm
         diameter spot to filter-paper, and allow to dry.

    5.   Apply 0.1 ml of furfuraldehyde solution to the spot, and allow to
         dry.

    6.   Expose the paper to concentrated hydrochloric acid fumes for 5
         minutes  in a fume cupboard.

    Results

         Meprobamate gives a black spot. Other carbamates, such as the
    carbamate pesticides, interfere in this test.

    Sensitivity

         Meprobamate, 100 mg/l.

    Confirmatory test

         Applicable to urine, stomach contents and scene residues.

    Reagents

    1.   Aqueous hydrochloric acid (1 mol/l).

    2.   Methanol: concentrated ammonium hydroxide (relative density 0.88)
         (100:1.5) (MA, see also section 6.73).

    3.   Furfuraldehyde (20 ml/l) in acetone, freshly prepared.

    4.   Concentrated sulfuric acid (relative density 1.83) solution
         (40 ml/l) in acetone, freshly prepared.

    5.   Van Urk reagent. Mix 1 g of  p-dimethylaminobenzaldehyde in
         100 ml of methanol with 10 ml of concentrated hydrochloric acid
         (relative density 1.18).

    6.   Silica gel thin-layer chromatography plates (10  20 cm, 20 m
         average particle size, see section 4.4.1).

    Standard

         Meprobamate 1 g/l in chloroform.

    Method

    1.   Add 1 ml of dilute hydrochloric acid and 10 ml of chloroform to
         10 ml of urine in a 30-ml glass tube.

    2.   Rotary-mix for 5 minutes, centrifuge for 5 minutes and discard
         the upper, aqueous phase.

    3.   Transfer the lower, organic layer to a 15-ml tapered glass tube
         and evaporate to dryness in a water-bath at 60C under a stream
         of compressed air or nitrogen.

    Thin-layer chromatography

    1.   Reconstitute the extract in 100 l of chloroform and spot two
         portions of 50 l of the extract and two portions of 10 l of the
         meprobamate standard on to separate columns of the plate.

    2.   Develop (10-cm run) using MA (saturated tank, see section 4.4.3)
         and dry until no smell of ammonia remains.

    3.   Spray one pair of columns (sample and standard) sequentially with
         (a) furfuraldehyde solution and (b) sulfuric acid solution, and
         allow to dry.

    4.   Spray the second pair of columns with van Urk reagent.

    Results

         Meprobamate (hRf, 10) gives a violet reaction with the
    furfuraldehyde/sulfuric acid spray, and a yellow colour with van Urk
    reagent. It may be necessary to redistill the furfuraldehyde, since
    this compound polymerizes on standing giving a brown discoloration.
    The sensitivity of the furfuraldehyde/sulfuric acid spray may be
    increased by gently heating the plate with a hair-drier after
    spraying.

         Other carbamate drugs, such as carisoprodol, mebutamate and
    tybamate, may interfere in this test, but meprobamate is by far the
    most common compound encountered.

    Sensitivity

         Meprobamate, 10 mg/l.

    Clinical interpretation

         Acute meprobamate poisoning may cause hypotension, hypothermia,
    muscle weakness, nystagmus, acidosis, coma, respiratory depression,
    pulmonary oedema, acute renal failure and disseminated intravascular
    coagulation. Treatment is normally symptomatic and supportive.

    6.68  Mercury

         Mercury (Hg) and its inorganic salts are used in the manufacture
    of thermometers, felt, paints, explosives, lamps, electrical equipment
    and batteries. Diethyl mercury, dimethyl mercury and a variety of
    other mercury compounds, including inorganic mercurials, are used as
    fungicides, primarily on seeds and bulbs, and in lawn sands. Mercuric
    chloride (HgCl2) is extremely toxic and ingestion of 1 g may prove
    fatal in an adult. As with antimony, arsenic and bismuth, mercury can
    be detected using the Reinsch test.

    Qualitative test

         Applicable to urine, stomach contents and scene residues. Reinsch
    test - see antimony monograph (section 6.5)

    Results

         Staining on the copper can be interpreted as follows:

    purple black - antimony

    dull black - arsenic

    shiny black - bismuth

    silver - mercury

         Selenium and tellurium may also give dark deposits, while high
    concentrations of sulfur may give a speckled appearance to the copper.

    Confirmatory test

         Applicable to silver-stained foil from the Reinsch test.

    Reagent

         Copper (I) iodide suspension. Dissolve 5 g of copper (II) sulfate
    and 3 g of ferrous sulfate in 10 ml of purified water with continuous
    stirring and add 7 g of potassium iodide in 50 ml of water. Allow the
    copper (I) iodide precipitate to form, filter and wash with water.
    Transfer to a brown glass bottle as a suspension with the aid of a
    little water. This suspension is quite stable.

    Method

         Add 0.1 ml of copper (I) iodide suspension to a filter-paper,
    place the foil on the suspension, cover and leave for 1-12 hours.

    Results

         A salmon-pink colour indicates the presence of mercury. Positive
    results may be obtained within 1 hour, but with low concentrations,
    colour development may take up to 12 hours.

    Sensitivity

         Mercury, 5 mg/l.

    Clinical interpretation

         Elemental mercury is poorly absorbed from the gastrointestinal
    tract and is not considered toxic by this route. Mercury vapour is
    absorbed through the skin and lungs and may give rise to stomatitis,
    increased salivation, a metallic taste, diarrhoea, pneumonitis and

    renal failure. The ingestion of mercuric salts may cause severe
    gastric pain, vomiting, bloody diarrhoea and also renal failure, which
    is usually the cause of death. Organomercurials are concentrated in
    the central nervous system and produce ataxia, chorea and convulsions.

         Treatment is symptomatic and supportive and may include chelation
    therapy. Mercury concentrations in blood and urine are good indicators
    of exposure, but only atomic absorption spectrophotometric methods of
    determination are reliable.

    6.69  Methadone

    ()-6-Dimethylamino-4,4-diphenylheptan-3-one; C21H27NO; relative
    molecular mass, 310

    CHEMICAL STRUCTURE 34

         Methadone is a narcotic analgesic structurally related to
    dextropropoxyphene and is widely used in the treatment of opioid
    dependence. Methadone is metabolized largely by  N-demethylation and
    hydroxylation. However, approximately 30% of an oral dose is excreted
    unchanged in urine. Plasma methadone concentrations attained on
    chronic (maintenance) therapy are very much higher than those
    associated with serious toxicity in patients not taking methadone
    chronically.

         There is no simple qualitative test for methadone, but this
    compound and its metabolites can be detected and identified by thin-
    layer chromatography of a basic solvent extract of urine (see section
    5.2.3).

    Clinical interpretation

         Acute overdosage with methadone may give rise to pin-point
    pupils, hypotension, hypothermia, coma, convulsions and pulmonary
    oedema. Death may ensue from profound respiratory depression. Naloxone
    rapidly reverses the central toxic effects of methadone (see section
    2.2.2).

    6.70  Methanol

    Methyl alcohol; wood alcohol; CH3OH; relative molecular mass, 32

         Methanol is used as a general and laboratory solvent, and in
    antifreeze (often with ethylene glycol), windscreen washer additives
    and duplicating fluids. In an adult, death may follow the ingestion of
    20-50 ml of methanol. Poisoning with industrial grades of ethanol
    (methylated spirit), which often contain methanol as a denaturant,
    also occurs, but is generally less serious than with methanol alone.
    This is because methanol toxicity results from its metabolism to
    formaldehyde and formate by alcohol dehydrogenase, a reaction
    inhibited by ethanol.

    Qualitative test

         Applicable to urine, stomach contents and scene residues.

    Reagent

         Potassium dichromate reagent. Potassium dichromate (25 g/l) in
    purified water:concentrated sulfuric acid (relative density 1.83)
    (1:1).

    Method

    1.   Apply 50 l of potassium dichromate reagent to a strip of glass-
         fibre filter-paper and insert the paper in the neck of a test-
         tube containing 1 ml of urine.

    2.   Lightly stopper the tube and place in a boiling water-bath for 2
         minutes.

    Results

         A change in colour from orange to green indicates the presence of
    volatile reducing agents such as methanol. However, other such
    compounds, for example ethanol, metaldehyde, and paraldehyde, also
    react.

    Sensitivity

         Methanol, 50 mg/l.

    Confirmatory test

         Applicable to urine, stomach contents and scene residues.

    Reagents

    1.   Potassium dichromate reagent. Potassium dichromate (25 g/l) in
         purified water: concentrated sulfuric acid (relative density
         1.83) (1:1).

    2.   Concentrated sulfuric acid (relative density 1.83).

    3.   Chromotropic acid (solid).

    Method

    1.   Add 0.1 ml of potassium dichromate reagent to 1 ml of urine and
         allow to stand at room temperature for 5 minutes.

    2.   Add 0.1 ml of ethanol and about 10 mg of chromotropic acid and
          gently add sulfuric acid down the side of the tube so that it
         forms a separate layer at the bottom.

    Results

         A violet colour at the junction of the two layers indicates the
    presence of methanol. Formaldehyde also gives a positive result in
    this test.

    Sensitivity

         Methanol, 50 mg/l.

    Clinical interpretation

         Acute methanol poisoning is characterized by delayed onset of
    coma, cyanosis, respiratory failure, marked metabolic acidosis,
    electrolyte imbalance, hyperglycaemia and blindness, which may be
    permanent. Treatment is aimed at correcting metabolic abnormalities,
    inhibiting methanol metabolism by giving ethanol and removing
    unchanged methanol by peritoneal dialysis or haemodialysis.
    Measurement of plasma ethanol concentrations can be useful in
    monitoring therapy with this compound (see section 6.46).

    6.71  Methaqualone

    2-Methyl-3- o-tolylquinazolin-4(3H)-one; C16H14N2O; relative
    molecular mass, 250

    CHEMICAL STRUCTURE 35

         Methaqualone is a nonbarbiturate hypnotic, but is now little used
    because of the risk of toxicity. Metabolic pathways include aromatic
    hydroxylation,  N-oxidation, and conjugation. Less than 2% of a dose
    is excreted unchanged. The estimated minimum lethal dose of
    methaqualone in an adult is 5 g.

         There is no simple qualitative test for methaqualone, but it can
    be detected and identified by thin-layer chromatography of a basic
    solvent extract of urine, stomach contents or scene residues (see
    section 5.2.3).

    Clinical interpretation

         Acute methaqualone poisoning may cause hypertonia, myoclonus,
    papilloedema, tachycardia, pulmonary oedema, coma and convulsions.
    Treatment is generally symptomatic and supportive. Charcoal
    haemoperfusion may be indicated in severe cases.

    6.72  Methyl bromide

         Methyl bromide (CH3Br) is used as a fumigant in ships' holds,
    grain silos and other large enclosed areas. Methyl bromide undergoes
    partial metabolism to give inorganic bromide  in vivo. Since the
    concentrations of this ion encountered in methyl bromide poisoning are
    much lower than in serious poisoning with inorganic bromides, it is
    thought that methyl bromide itself is the primary toxin.

         The qualitative test given below serves to indicate the presence
    of inorganic bromides or iodides, and the appropriate confirmatory
    test must then be used or blood bromide measured. There is no simple
    method for measuring unchanged methyl bromide.

    Qualitative test

         Applicable to urine. Detects inorganic bromide.

    Reagents

    1.   Aqueous nitric acid (2 mol/l).

    2.   Aqueous silver nitrate solution (10 g/l).

    3.   Concentrated ammonium hydroxide (relative density 0.88).

    Method

    1.   Add 0.1 ml of nitric acid to 1 ml of clear test solution, mix for
         5 seconds and add 0.1 ml of silver nitrate solution.

    2.   Centrifuge to isolate any significant precipitate and treat with
         0.1 ml of concentrated ammonium hydroxide.

    Results

         A white precipitate soluble in ammonium hydroxide indicates
    chloride, an off-white precipitate sparingly soluble in ammonium
    hydroxide indicates the presence of bromide and a creamy-yellow,
    insoluble precipitate indicates iodide.

    Sensitivity

         Bromide, 50 mg/l.

    Confirmatory test

         Applicable to urine. Detects inorganic bromide.

    Reagents

    1.   Saturated fluorescein solution in aqueous acetic acid (600 ml/l).

    2.   Concentrated sulfuric acid (relative density 1.83).

    3.   Potassium permanganate (solid)

    Method

    1.   Soak a strip of filter-paper in fluorescein solution.

    2.   Add about 50 mg of potassium permanganate to 2 ml of test
         solution in a 10-ml test-tube.

    3.   Add 0.2 ml of concentrated sulfuric acid and hold the
         fluorescein-impregnated filter-paper in the mouth of the tube.

    Results

         The bromide is oxidized to free bromine. This reacts with the
    yellow dye fluorescein to give eosin (tetrabromofluorescein) which has
    a pink/red colour.

    Sensitivity

         Bromide, 50 mg/l.

    Quantitative assay

         Applicable to plasma or serum (2 ml).

    Reagents

    1.   Aqueous chloroauric acid. Dissolve 0.5 g of chloroauric acid
         (gold chloride, HAuCl4xH2O) in 100 ml of purified water.

    2.   Aqueous trichloroacetic acid (200 g/l).

    Standards

         Dissolve 1.288 g of sodium bromide in 500 ml of purified water
    (bromide ion 2 g/l). Prepare serial dilutions in purified water
    containing bromide ion concentrations of 0.2, 0.4, 0.6, 0.8, 1.2 and
    1.6 g/l.

    Method

    1.   Add 6 ml of trichloroacetic acid solution to 2 ml of sample in a
         10-ml test-tube, vortex-mix for 30 seconds and allow to stand for
         15 minutes.

    2.   Centrifuge in a bench centrifuge for 5 minutes and filter the
         supernatant through phase-separating filter-paper into a clean
         tube.

    3.   Add 1 ml of chloroauric acid solution to 4 ml of the clear
         supernatant and vortex-mix for 5 seconds.

    4.   Record the absorbance at 440 nm against a purified water blank
         (see section 4.5.2).

    Results

         Construct a calibration graph of bromide concentration against
    absorbance by analysis of the standard bromide solutions, and
    calculate the concentration of bromide ion in the sample. The
    calibration is linear for concentrations from 25 mg/l to 2.5 g/l. This
    method is not reliable with specimens that may give turbid
    supernatants, e.g. postmortem samples.

    Sensitivity

         Bromide, 25 mg/l.

    Clinical interpretation

         Symptoms of poisoning due to methyl bromide often develop several
    hours after exposure and include confusion, dizziness, headache,
    nausea, vomiting, abdominal pain, blurred vision, hyporeflexia and
    paraesthesia. Coma and convulsions may occur in severe cases, and
    pulmonary oedema, jaundice and oliguria have also been described.
    Treatment is symptomatic and supportive.

         Normal serum bromide concentrations are less than 10 mg/l. After
    the therapeutic administration of inorganic bromides, concentrations
    of up to 80 mg/l may be attained; toxicity is usually associated with
    concentrations greater than 500 mg/l. On the other hand, blood bromide
    concentrations of 90-400 mg/l have been reported in fatal methyl
    bromide poisoning.

    6.73  Morphine

    (4a R,5 S,7a R,SR,9c S)-4a,5,7a,8,9,9c-Hexahydro-12-methyl-8,9c-
    imino-ethanophenanthro[4,5-bcd]furan-3,5-diol monohydrate;
    C17H19NO3H2O; relative molecular mass, 303

    CHEMICAL STRUCTURE 36

         Morphine is the principal alkaloid of opium and is a potent
    narcotic analgesic. Diamorphine (heroin, 3,6- O-diacetylmorphine) has
    two to three times the potency of morphine and is obtained by treating
    morphine (or opium in the case of illicit preparations) with acetic
    anhydride.

         Diamorphine is rapidly hydrolysed  in vivo to 6-acetylmorphine
    and thence to morphine. Morphine is also a metabolite of codeine.
    Approximately 5% of a dose of morphine is metabolized to normorphine,
    but conjugation with glucuronic acid is the major pathway. The
    principal product is morphine-3-glucuronide, but morphine-6-
    glucuronide is also formed. Free morphine in urine accounts for about
    10% of a dose, while morphine-3-glucuronide accounts for 75%. The
    estimated minimum fatal dose of morphine or diamorphine in an adult
    unaccustomed to taking these compounds is 100-200 mg.

         Morphine can be detected by thin-layer chromatography of an
    extract of urine, stomach contents, or scene residues at pH 8.5-9.
    Since a large proportion of a dose of diamorphine or morphine is
    excreted as glucuronides, prior hydrolysis of urine can increase the
    sensitivity of the procedure.

    Qualitative test

         Applicable to urine.

    Reagents

    1.   Concentrated hydrochloric acid (relative density 1.18).

    2.   Sodium bicarbonate (solid).

    3.   Silica gel thin-layer chromatography plate (10  20 cm, 20 m
         average particle size; see section 4.4.1).

    4.   Methanol:concentrated ammonium hydroxide (relative density 0.88)
         (99:1.5) (MA).

    5.   Ethyl acetate:methanol:concentrated ammonium hydroxide (relative
         density 0.88) (85:10:5) (EMA).

    6.   Iodoplatinate reagent. Mix 0.25 g of platinic chloride and 5 g of
         potassium iodide in 100 ml of purified water.

    Standards

    1.   Hydrolysis standards: codeine, dihydrocodeine and morphine-3-
         glucuronide (all 1 mg/l) in blank urine.

    2.   Thin-layer chromatography standards: cocaine, codeine,
         dihydrocodeine, methadone and morphine (all 1 g/l) in chloroform.

    Method

    1.   Add 2 ml of concentrated hydrochloric acid to 10 ml of urine or
         hydrolysis standard in a test-tube.

    2.   Heat on a boiling water-bath for 30 minutes.

    3.   Allow to cool, decant into a 250 ml beaker and  slowly add
         sodium bicarbonate until effervescence ceases and solid sodium
         bicarbonate remains in the beaker.  Take care - this reaction
          can be violent.

    4.   Decant the hydrolysate into a clean tube, add 10 ml of ethyl
         acetate:propan-2-ol (9:1) and vortex-mix for 3 minutes.

    5.   Centrifuge for 5 minutes, filter the upper organic phase through
         phase-separating filter-paper into a clean tube and discard the
         lower, aqueous layer.

    6.   Evaporate the extract to dryness under a stream of compressed
         air.

    Thin-layer chromatography

    1.   Reconstitute the extracts in 50 l of ethyl acetate:propan-2-ol
         (9:1) and spot equal portions of the sample and standard
         extracts, and 10 l of the chromatography standard on three
         columns of two plates.

    2.   Develop one plate in MA and the second in EMA (10-cm run,
         saturated tanks, see section 4.4.3).

    3.   Allow to dry and ensure that no smell of ammonia remains before
         spraying both plates with iodoplatinate reagent.

    Results

         The hRf values and colour reactions of the drugs in the standard
    mixtures and some additional compounds of interest are given in Table
    30. This procedure gives no information as to the presence of
    diamorphine since this compound and both 3- O-acetylmorphine and
    6- O-acetylmorphine, as well as morphine glucuronides, are hydrolysed
    to morphine.

         The hydrolysis standard extract must be analysed as well as the
    chromatography standard, since compounds extracted from hydrolysed
    urine tend to migrate more slowly on thin-layer chromatography owing
    to the influence of co-extracted compounds, and this must be allowed
    for in the interpretation of results.

         It is important to ensure that only concentrated ammonium
    hydroxide (relative density 0.88) is used to prepare the mobile phases
    (MA and EMA) and that a given batch of eluent is used only three times
    for MA and five times for EMA, as discussed in section 5.2.3.

    Table 30.  Thin-layer chromatography of morphine and related
               compounds: hRf values and colour reactions
                                                                        

    Compound                           hRf           Colour
                                                
                                       EMA    MA
                                                                        

    Methadone metabolite               78     22     Purple
    Cocainea                           77     73     Violet
    Methadone                          77     56     Purple (white ring)
    Pentazocine                        72     71     Speckled blue
    Cyclizine                          68     68     Speckled dark blue
    Dextropropoxyphene metabolitea     66     55     Blue (streaks
                                                     on MA)
    Pethidine                          62     61     Purple
    Nicotine                           61     68     Dark brown
    Chloroquine                        52     44     Dark blue
    Quinine                            52     70     Speckled blue
    6-Acetylmorphinea                  48     53     Speckled blue
    Cotinine (from nicotine)           40     70     Khaki
    Hydroxychloroquine                 38     53     Blue
    Codeine                            35     47     Blue
    Norpethidine                       34     33     Purple (white rim)
    (from pethidine)
    Dihydrocodeine                     27     37     Dark blue
    Norcotinine                        23     70     Khaki
    (from cotinine)
    Desethylchloroquine                22     22     Dark blue
    Morphine                           20     47     Blue-black
    Codeine metabolite                 18     25     Blue-purple
    Dihydrocodeine metabolite          16     --     Dark blue
                                                                        

    a    Not normally present in urine hydrolysates.

    Sensitivity

         Morphine (free + conjugated), 1 mg/l.

    Clinical interpretation

         Acute poisoning with morphine and diamorphine gives rise to
    pin-point pupils, hypotension, hypothermia, coma, convulsions and
    pulmonary oedema. Death may ensue from profound respiratory
    depression. Naloxone rapidly reverses the central toxic effects of
    morphine (see section 2.2.2).

    6.74  Nicotine

    3-(1-Methylpyrrolidin-2-yl)pyridine; C10H14N2; relative molecular
    mass, 162

    CHEMICAL STRUCTURE 37

         Nicotine is an alkaloid derived from the leaves of  Nicotiana 
     tabacum. As little as 40 mg of nicotine can prove fatal in an adult.
    Nicotine is commonly encountered in tobacco although usually in
    concentrations insufficient to cause acute poisoning, except when
    ingested by young children. Nicotine occurs in higher concentrations
    in some herbal medicines, and is also used as a fumigant in
    horticulture. Nicotine can be absorbed rapidly through the skin, and
    is metabolized principally by  N-demethylation to give cotinine.

         There is no simple qualitative test for nicotine, but this
    compound and cotinine can be detected and identified by thin-layer
    chromatography of a basic solvent extract of urine (see section
    5.2.3).

    Clinical interpretation

         Initially nausea, dizziness, vomiting, respiratory stimulation,
    headache, tachycardia, sweating and excessive salivation occur
    followed by collapse, convulsions, cardiac arrhythmias and coma in
    severe cases. Death may supervene rapidly or be delayed by several
    hours. Treatment is symptomatic and supportive.

    6.75  Nitrates

         Nitrates such as sodium nitrate (NaNO3) are most commonly found
    in inorganic fertilizers, but are also used as antiseptics, food
    preservatives and explosives. Death in an adult may follow the
    ingestion of about 15 g of the sodium or potassium salt. Organic
    nitrates, such as glyceryl trinitrate, are used as vasodilators.
    Nitrates are metabolized to nitrites in the gastrointestinal tract.
    Nitrates are strong oxidizing agents and the test given below will
    also detect compounds with similar properties, such as bromates,
    chlorates, hypochlorites, iodates and nitrites.

    Qualitative test

         Applicable to stomach contents and scene residues.

    Reagent

         Diphenylamine (10 g/l) in concentrated sulfuric acid (relative
    density 1.83).

    Method

    1.   Filter, if necessary, 5 ml of stomach contents into a 10-ml glass
         tube.

    2.   Add 0.5 ml of filtrate or scene residue to a clean tube and
         slowly add 0.5 ml of diphenylamine solution down the side of the
         tube so that it forms a layer under the sample.

    Results

         A true positive is indicated by a strong blue colour which
    develops immediately at the junction of the two layers. A light blue
    colour will be given by most samples of stomach contents owing to the
    presence of organic material. Since all strong oxidizing agents are
    rapidly reduced in biological samples, the test should be performed as
    soon as possible after receipt of the sample.

    Sensitivity

         Nitrate, 10 mg/l.

    Confirmatory test

         Applicable to urine, stomach contents and scene residues.

    Reagents

    1.   Sulfamic acid reagent. Mix 1 ml of ammonium sulfamate (150 g/l)
         and 1 ml of aqueous hydrochloric acid (2 mol/l), freshly
         prepared.

    2.   Aqueous imipramine hydrochloride solution (20 g/l).

    3.   Concentrated sulfuric acid (relative density 1.83).

    Method

    1.   Mix 0.1 ml of test solution and 0.1 ml of sulfamic acid reagent.

    2.   Add 0.1 ml of imipramine solution, and  carefully add 0.2 ml of
         concentrated sulfuric acid down the side of the tube so that it
         forms a layer underneath the test mixture.

    Results

         An intense blue colour at the junction of the two layers confirms
    nitrate. Sulfamic acid treatment is used to remove any nitrite present
    prior to the test.

    Sensitivity

         Nitrate, 50 mg/l.

    Clinical interpretation

         Acute poisoning with nitrates can cause nausea, vomiting,
    diarrhoea, abdominal pain, confusion, coma and convulsions. In
    addition, nitrates may give rise to headache, flushing, dizziness,
    hypotension and collapse. Treatment is symptomatic and supportive.
    Methaemoglobinaemia is often produced and this may be indicated by
    dark chocolate-coloured blood (see section 3.2.2). Blood
    methaemoglobin can be measured but is unstable and the use of stored
    samples is unreliable.

    6.76  Nitrites

         Nitrites such as sodium nitrite (NaNO2) were formerly used as
    vasodilators, and are used to prevent rusting, as food preservatives
    and in explosives. Nitrites may also arise from the metabolism of
    nitrates. The fatal dose of sodium nitrite is about 10 g, although

    ingestion of as little as 2 g has caused death in an adult. Nitrites
    are strong oxidizing agents and the test given below will also detect
    compounds with similar properties such as bromates, chlorates,
    hypochlorites, iodates and nitrates.

    Qualitative test

         Applicable to stomach contents and scene residues.

    Reagent

         Diphenylamine (10 g/l) in concentrated sulfuric acid (relative
    density 1.83).

    Method

    1.   Filter, if necessary, 5 ml of stomach contents into a 10-ml glass
         tube.

    2.   Add 0.5 ml of filtrate or scene residue to a clean tube and
         slowly add 0.5 ml of diphenylamine solution down the side of the
         tube so that it forms a layer under the sample.

    Results

         A true positive is indicated by a strong blue colour which
    develops immediately at the junction of the two layers. A light blue
    colour will be given by most samples of stomach contents owing to the
    presence of organic material. Since all strong oxidizing agents are
    rapidly reduced in biological samples, the test should be performed as
    soon as possible after receipt of the sample.

    Sensitivity

         Nitrite, 10 mg/l.

    Confirmatory tests

         Applicable to urine, stomach contents and scene residues.

    1.  Imipramine/hydrochloric acid test

    Reagents

    1.   Aqueous imipramine hydrochloride (20 g/l).

    2.   Concentrated hydrochloric acid (relative density 1.18).

    Method

         To 0.1 ml of imipramine solution add 0.1 ml of test solution and
    0.2 ml of hydrochloric acid.

    Results

         A blue colour is specific for nitrite.

    Sensitivity

         Nitrite, 1 mg/l.

    2.  Sulfanilic acid/1-aminonaphthalene test

    Reagents

    1.   Sulfanilic acid (10 g/l) in aqueous acetic acid (300 ml/l).

    2.   1-Aminonaphthalene (1 g/l) in aqueous acetic acid (300 ml/l).

    Method

    1.   Add 0.1 ml of test solution to 0.1 ml of sulfanilic acid
         solution.

    2.   Mix and add 0.1 ml of 1-aminonaphthalene solution.

    Results

         A purple/red colour is specific for nitrite. If the test solution
    is strongly acidic or basic the pH should be adjusted beforehand to
    about 7 (universal indicator paper) by carefully adding 2 mol/l
    aqueous hydrochloric acid or sodium hydroxide.

    Sensitivity

         Nitrite, 0.2 mg/l.

    Quantitative assay

         Applicable to urine.

    Reagents

    1.   Aqueous sodium acetate solution (164 g/l).

    2.   Sulfanilic acid (6 g/l) in concentrated hydrochloric acid
         (relative density 1.18):purified water (1:4).

    3.   1-Aminonaphthalene (4.8 g/l) in concentrated hydrochloric acid
         (relative density 1.18):methanol (1:4).

    Standards

         Solutions containing nitrite ion at concentrations of 0, 10, 20,
    50 and 100 mg/l in purified water, prepared by dilution from aqueous
    sodium nitrite solution (1.50 g/l, equivalent to a nitrite ion
    concentration of 1.00 g/l).

    Method

    1.   Mix 0.5 ml of sample or standard and 0.5 ml of sulfanilic acid
         solution in a 25-ml volumetric flask.

    2.   Allow to stand for 10 minutes, add 0.5 ml of 1-aminonaphthalene
         solution and 0.5 ml of sodium acetate solution and dilute to
         25 ml with purified water.

    3.   Allow to stand for 10 minutes and measure the absorbance at
         510 nm against a water blank carried through the procedure (see
         section 4.5.2).

    Results

         Construct a graph of absorbance against nitrite concentration by
    analysis of the standard solutions and calculate the nitrite
    concentration in the sample.

    Sensitivity

         Nitrite, 5 mg/l.

    Clinical interpretation

         Acute poisoning with nitrites may cause nausea, vomiting,
    diarrhoea, abdominal pain, confusion, coma and convulsions. In
    addition, nitrites may give rise to headache, flushing, dizziness,
    hypotension and collapse. Methaemoglobinaemia is often produced and
    this may be indicated by dark chocolate-coloured blood (see section
    3.2.2). Blood methaemoglobin can be measured but is unstable and the
    use of stored samples is unreliable. Treatment is symptomatic and
    supportive. Urinary nitrite ion concentrations of 10 mg/l and above
    have been reported in fatalities.

    6.77  Nitrobenzene

    Nitrobenzene (nitrobenzol; C6H5NO2; relative molecular mass, 123)
    has a characteristic odour of bitter almonds and is used in the
    manufacture of aniline, as a solvent for cellulose ethers, in metal
    and shoe polishes, perfumes, dyes and soaps, and as a synthetic
    intermediate. The acute toxicity of nitrobenzene is very similar
    to that of aniline, probably because of metabolic conversion.
    Nitrobenzene is metabolized to  p-aminophenol and  N-acetyl-
     p-aminophenol (paracetamol), which are both excreted in urine as
    sulfate and glucuronide conjugates. On hydrolysis of urine,
     p-aminophenol is reformed and can be detected using the
     o-cresol/ammonia test.

    Qualitative test

         Applicable to urine.  o-Cresol/ammonia test - see paracetamol
    monograph (section 6.83).

    Results

         A strong, royal blue colour developing immediately indicates the
    presence of  p-aminophenol. Metabolites of paracetamol (and of
    phenacetin) also give  p-aminophenol on hydrolysis and thus
    interfere. Ethylenediamine (from aminophylline, for example - see
    section 6.105) gives a green colour in this test.

    Sensitivity

     p-Aminophenol, 1 mg/l.

    Clinical interpretation

         Poisoning with nitrobenzene may arise from inhalation or dermal
    absorption as well as ingestion. Symptoms occur within 1-3 hours of
    exposure and include confusion, nausea, vomiting and diarrhoea, with
    convulsions, coma and hepatorenal damage in severe cases. Haemolysis,
    red (wine-coloured) urine, and methaemoglobinaemia (dark chocolate-
    coloured blood) are characteristic features of poisoning with
    nitrobenzene, as with aniline (see section 3.2.2).

         Blood methaemoglobin can be measured, but is unstable and the use
    of stored samples is unreliable. However, hepatic and renal function
    tests are essential. Treatment may include intravenous methylene blue,
    but this is contraindicated in patients with glucose-6-phosphate
    dehydrogenase deficiency, since there is a high risk of inducing
    haemolysis.

    6.78  Nortriptyline

    3-(10,11-Dihydro-5 H-dibenzo [a,d]cyclohepten-5-ylidene)-
     N-methylpropylamine; C19H21N; relative molecular mass, 263

    CHEMICAL STRUCTURE 38

         Nortriptyline is the  N-demethylated metabolite of amitriptyline
    and is also a tricyclic antidepressant in its own right.

         There is no simple test for nortriptyline, but this compound and
    other tricyclic antidepressants can be easily detected and identified
    by thin-layer chromatography of a basic solvent extract of urine,
    stomach contents or scene residues (see section 5.2.3).

    Clinical interpretation

         Acute poisoning with nortriptyline and other tricyclic
    antidepressants may be associated with dilated pupils, hypotension,
    hypothermia, cardiac arrhythmias, depressed respiration, coma,
    convulsions and cardiorespiratory arrest. Urinary retention is also a
    feature of poisoning with these compounds and this may delay
    procurement of an appropriate specimen for analysis.

         Treatment is generally symptomatic and supportive. The use of
    antiarrhythmic agents is generally avoided, but alkalinization using
    sodium bicarbonate is sometimes employed. Quantitative measurements in
    blood are not normally required for management.

    6.79  Organochlorine pesticides

         These compounds are chlorinated hydrocarbons of diverse
    structure. Some that may be encountered are listed in Table 31. In
    addition, benzene hexachloride (BHC) is a mixture of several
    hexachlorocyclohexane isomers.

    Table 31.  Some organochlorine pesticides
                                                                        

    Compound       Chemical name                               Relative
                                                               molecular
                                                                 mass
                                                                        

    Aldrin         1,2,3,4,10,10-Hexachloro-1,4,4a,5,8,8a-       365
      (HHDN)       hexahydro-1,4:5,8-dimethanonaphthalene
    Chlordane      1,2,4,5,6,7,8,8-Octachloro-2,3,3a,4,7,7a-     410
                   hexahydro-4,7-methanoindene
    DDT            Principally 1,1,1-trichloro-2,2-bis-          355
                   (4-chlorophenyl)ethane
    Dieldrin       1,2,3,4,10,10-Hexachloro-6,7-epoxy-1,4,4a,5,  381
      (HEOD)       6,7,8,8a-octahydro-exo-1,4-endo-5,8-
                   dimethanonaphthalene
    Endrin         1,2,3,4,10,10-Hexachloro-6,7-epoxy-1,4,4a,5,  381
                   6,7,8,8a-octahydro-endo-1,4-endo-5,8-
                   dimethanonaphthalene
    Heptachlor     1,4,5,6,7,8,8-Heptachloro-3a,4,7,7a-          373
                   tetrahydro-4,7-methanoindene
    Lindane        1-alpha,2-alpha,3,4-alpha,5-alpha,6-        291
      (gamma-HCH)  Hexachlorocyclohexane
                                                                        

         These compounds are commonly used as insecticides in many
    countries, and persist in the environment. Aldrin, dieldrin and endrin
    (approximate acute lethal dose, 5 g in an adult) are more toxic than
    lindane or DDT (approximate lethal dose, 30 g).

         There are no reliable simple tests for these compounds, although
    a qualitative analysis can be performed by thin-layer chromatography
    of a solvent extract of the specimen.

    Qualitative test

         Applicable to stomach contents and scene residues.

    Reagents

    1.   Petroleum ether (40-60C boiling fraction).

    2.   Aqueous sodium hydroxide solution (20 g/l).

    3.   Sodium sulfate (anhydrous).

    4.   Aqueous potassium permanganate (0.1 mol/l).

    5.   2-Aminoethanol (ethanolamine).

    6.   Silver nitrate reagent. Aqueous silver nitrate (0.1
         mol/l):concentrated nitric acid (relative density 1.42) (10:1).

    7.   Silica gel thin-layer chromatography plate (5  20 cm, 20 m
         average particle size; see section 4.4.1).

    Standards

         Aldrin, lindane, dicophane and heptachlor (all 1 g/l) in
    methanol.

    Method

    1.   Extract 10 ml of sample with 5 ml of petroleum ether for 5
         minutes using a rotary mixer.

    2.   Allow to stand for 5 minutes, take off the upper, ether layer and
         re-extract with a second 5-ml portion of petroleum ether.

    3.   Combine the ether extracts and wash with 5-ml portions of:

         (a) purified water;

         (b) sodium hydroxide solution;

         (c) purified water.

    4.   Filter the extract through phase-separating filter-paper into a
         clean tube, dry over about 5 g of sodium sulfate and evaporate to
         dryness under a stream of compressed air or nitrogen.

    Thin-layer chromatography

    1.   Reconstitute the extract in 100 l of methanol and spot 20 l on
         to a column on the plate.

    2.   Spot 10 l of the standard mixture on a second column.

    3.   Develop the chromatogram (10-cm run) using cyclohexane (saturated
         tank; see section 4.4.3) and allow to dry.

    4.   Spray the plate with potassium permanganate solution, spray
         lightly with 2-aminoethanol and heat (preferably in an oven) at
         100C for 20 minutes.

    5.   Allow to cool, spray with silver nitrate reagent and expose to
         ultraviolet light (254 nm) for 15 minutes.

    Results

         The compounds of interest give brown/black spots. Identification
    is by comparison with the standard chromatogram. Dieldrin and endrin
    are not detected under the conditions used. Approximate hRf values
    for the remaining compounds are as follows:

    lindane 09

    dicophane 26

    heptachlor 34

    aldrin 41

    Sensitivity

         Organochlorine pesticide, 2.5 mg/l (aldrin 10 mg/l).

    Clinical interpretation

         Features of poisoning with organochlorine pesticides include
    vomiting, weakness and numbness of the extremities, apprehension,
    excitement, diarrhoea and muscular tremor, with convulsions and
    respiratory depression in severe cases. Treatment is symptomatic and
    supportive.

    6.80  Organophosphorus pesticides

         This is a very large group of compounds. There are four basic
    structures:

    CHEMICAL STRUCTURE 39

    where R = alkyl; X = a wide variety of structures.

         An example from each group is given in Table 32. Some
    organophosphorus pesticides are used as herbicides and are relatively
    nontoxic to humans. However, most are insecticides which interfere
    with neurotransmission by inhibition of acetylcholinesterase. This
    property forms the basis of the confirmatory test described below.
    Organophosphorus pesticides often have a pungent smell (of garlic),
    and this property can be helpful in indicating the diagnosis.

        Table 32.  Some organophosphorus pesticides
                                                                              

    Compound    Chemical name                                        Relative
                                                                     molecular
                                                                       mass
                                                                              

    Malathion   Diethyl-2-(dimethoxyphosphinothioylthio)               330
                  succinate
    Parathion   O,O-Diethyl-O-4-nitrophenylphosphorothioate            291
    Omethoate   O,O-Dimethyl-S-methylcarbamoylmethylphosphorothioate   213
    Mevinphos   2-Methoxycarbonyl-1-methylvinyldimethyl                224
                  phosphate
                                                                              
    
         Many of these compounds are hydrolysed in alkaline solution while
    some (for example azinphos-methyl, diazinon, and malathion) are also
    unstable in acid. For this reason it is important to adjust the pH of
    stomach contents and scene residues to about 7 prior to the analysis.

    Qualitative test

         Applicable to stomach contents and scene residues.

    Reagents

    1.   Sodium bicarbonate (solid).

    2.   Cyclohexane:acetone:chloroform (70:25:5).

    3.   Acetone:tetraethylenepentamine (9:1).

    4.   4-( p-Nitrobenzyl)pyridine (20 g/l) in acetone:
         tetraethylenepentamine (9:1).

    5.   Silica gel thin-layer chromatography plate (5  20 cm, 20 m
         average particle size; see section 4.4.1).

    Standards

         Dimethoate, methidathion, dioxathion and chlorpyrifos (all 1 g/l)
    in methanol.

    Method

    1.   If necessary, carefully adjust the pH of 10 ml of sample to about
         7 by adding solid sodium bicarbonate.

    2.   Extract 10 ml of sample with 5 ml of methyl tertiary-butyl ether
         for 5 minutes using a rotary mixer.

    3.   Allow to stand for 5 minutes, take off the upper, ether layer and
         re-extract with a second 5-ml portion of methyl tertiary-butyl
         ether.

    4.   Combine the extracts, filter through phase-separating filter-
         paper into a clean tube and evaporate to dryness under a stream
         of compressed air or nitrogen.

    Thin-layer chromatography

    1.   Reconstitute the extract in 100 l of methanol and spot 20 l on
         a column marked on the plate.

    2.   Spot 10 l of the standard mixture on a second column.

    3.   Develop the chromatogram (10-cm run) using cyclohexane:
         acetone:chloroform (saturated tank, see section 4.4.3) and allow
         to dry.

    4.   Spray the plate with 4-( p-nitrobenzyl)pyridine solution and
         heat, preferably in an oven, at 110C for 30 minutes.

    5.   Allow to cool and spray with acetone:tetraethylenepentamine
         (9:1).

    Results

         The compounds of interest give purple spots on a pale brown
    background. Approximate hRf values are as follows:

    dimethoate          11

    methidathion        40

    malathion           42

    dioxathion          47

    propetamphos        49

    bromophos           54

    chlorpyrifos        58

    Sensitivity

         Organophosphorus pesticide, 5 mg/l.

    Confirmatory test

         Applicable to plasma or serum. See cholinesterase activity
    monograph (section 6.30).

    Results

         The presence of an acetylcholinesterase inhibitor is indicated if
    the yellow colour in the control tube is deeper than that in the test
    tube. If the colour in the tube containing pralidoxime is similar to
    that in the control tube, this provides further confirmation that an
    inhibitor of acetylcholinesterase is present in the sample. Of course,
    other inhibitors of acetylcholinesterase, such as many carbamate
    pesticides, also give a positive result in this test.

    Clinical interpretation

         Exposure to organophosphorus insecticides may cause
    bronchorrhoea, respiratory distress, excessive salivation, nausea,
    muscle weakness and eventually paralysis. Measurement of erythrocyte
    cholinesterase activity (see section 3.1.5) provides a method of
    assessing the severity of poisoning with this group of compounds.
    Treatment is symptomatic and supportive, and may include the
    administration of atropine and pralidoxime (see Table 4).

    6.81  Orphenadrine

     N,N-Dimethyl-2-(2-methylbenzylhydryloxy)ethylamine; C18H23NO;
    relative molecular mass, 269.

    CHEMICAL STRUCTURE 40

         Orphenadrine is an anticholinergic agent used in the treatment
    of parkinsonism. Up to 60% of an oral dose is excreted in urine
    within three days. Metabolism is by  N-demethylation to give
     N-desmethylorphenadrine and  N,N-didesmethylorphenadrine,
     N-oxidation to give orphenadrine  N-oxide, and a number of other
    pathways. The fatal dose of orphenadrine in an adult is thought to be
    2-4 g.

         There is no simple qualitative test for orphenadrine, but it can
    be detected and identified by thin-layer chromatography of a basic
    solvent extract of urine, stomach contents or scene residues (see
    section 5.2.3).

    Clinical interpretation

         Acute orphenadrine poisoning may cause a dry mouth, nausea,
    vomiting, tachycardia, hyperthermia, dizziness, excitement, confusion,
    hallucinations and convulsions. Treatment is symptomatic and
    supportive.

    6.82  Oxalates

         Oxalates are used in bleaching and cleaning agents, metal
    polishes and anti-rust agents. Oxalic acid ((COOH)22H2O; relative
    molecular mass, 126) also occurs in several plants, the leaves of
    domestic rhubarb and other members of the dock family (Polygonaceae)
    containing a particularly high concentration. The fatal dose of oxalic
    acid in an adult is of the order of 10 g. Oxalic acid is also a major
    toxic metabolite of ethylene glycol.

    Qualitative test

         Applicable to urine, stomach contents and scene residues.

    Reagents

    1.   Aqueous calcium chloride solution (100 g/l).

    2.   Aqueous acetic acid (300 ml/l).

    3.   Aqueous hydrochloric acid (2 mol/l).

    Method

    1.   Mix 1 ml of calcium chloride solution and 2 ml of clear test
         solution.

    2.   If a precipitate forms, add 1 ml of acetic acid.

    3.   If the precipitate remains, isolate by centrifugation and add 1
         ml of dilute hydrochloric acid.

    Results

         A white precipitate, insoluble in acetic acid, indicates oxalate,
    fluoride or sulfate. If the precipitate, once isolated, is soluble in
    dilute hydrochloric acid (step 3), oxalate is indicated.

    Sensitivity

         Oxalate, 250 mg/l.

    Confirmatory tests

    1.  Applicable to the precipitate from the qualitative test above.

    Reagents

    1.   Urea (solid).

    2.   Thiobarbituric acid (solid) ( not thiopental - see section 6.9).

    Method

    1.   Wash the precipitate twice with purified water, wash with acetone
         and dry at room temperature.

    2.   Suspend the precipitate in 50 l of methanol in a micro-test-tube
         and add about 20 mg of urea and about 200 mg of thiobarbituric
         acid.

    3.   Mix thoroughly and heat gently on a micro-burner to 140-160C.

    Results

         The rapid formation of a bright orange-red product, soluble in
    methanol, confirms oxalate.

    Sensitivity

         Oxalate, 250 mg/l.

    2.  Applicable to stomach contents and scene residues.

    Reagents

    1.   Concentrated ammonium hydroxide (relative density 0.88).

    2.   Thiobarbituric acid (solid) ( not thiopental - see section 6.9).

    Method

    1.   Mix 50 l of test solution with 100 l of concentrated ammonium
         hydroxide in a micro-test-tube and carefully evaporate to dryness
         using a micro-burner.

    2.   Add about 200 mg of thiobarbituric acid and gently reheat to
         140-160C.

    Results

         The rapid formation of a bright orange-red product, soluble in
    methanol, confirms oxalate.

    Sensitivity

         Oxalate, 250 mg/l.

    Clinical interpretation

         In addition to irritant effects on the alimentary tract when
    ingested, oxalate sequesters calcium, causing hypocalcaemia, muscular
    twitching and eventually tetany, convulsions, flank pain, acute renal
    failure and cardiac arrest. The crystalluria produced may be
    diagnostic (section 5.2.1). Treatment is normally symptomatic and
    supportive.

    6.83  Paracetamol

    Acetaminophen;  N-acetyl- p-aminophenol; C8H9NO2; relative
    molecular mass, 151

    CHEMICAL STRUCTURE 41

         Paracetamol is a widely used analgesic and sometimes occurs in
    combination with other drugs such as dextropropoxyphene. It is a
    metabolite of phenacetin and of benorilate, and is itself largely
    metabolized by conjugation with glucuronic acid and sulfate prior to
    urinary excretion.

         Hydrolysis of the glucuronate and sulfate conjugated with
    concentrated hydrochloric acid gives  p-aminophenol, which can be
    conjugated with  o-cresol to form a strongly coloured dye, thus
    giving a sensitive qualitative test. Protein precipitation with
    trichloroacetic acid and subsequent treatment with nitrous acid and
    spectrophotometric measurement of the nitrated derivative give a
    selective assay for paracetamol in plasma.

    Qualitative test

         Applicable to urine, stomach contents and scene residues.

    Reagents

    1.   Concentrated hydrochloric acid (relative density 1.18)

    2.   Aqueous  o-cresol solution (10 g/l).

    3.   Aqueous ammonium hydroxide solution (4 mol/l).

    Method

    1.   Add 0.5 ml of hydrochloric acid to 0.5 ml of sample, boil for 10
         minutes and cool.

    2.   Add 1 ml of  o-cresol solution to 0.2 ml of the hydrolysate.

    3.   Add 2 ml of ammonium hydroxide solution and mix for 5 seconds.

    Results

         A strong, royal blue colour developing immediately indicates the
    presence of paracetamol. This test is very sensitive and will detect
    therapeutic dosage with paracetamol 24-48 hours later.

         Only aromatic amines, such as aniline, which also give rise to
     p-aminophenol in urine after hydrolysis are known to interfere.
    Ethylenediamine (from aminophylline, for example; see section 6.105)
    gives a green colour in this test.

    Sensitivity

          p-Aminophenol, 1 mg/l.

    Quantitative assay

         Applicable to plasma or serum.

    Reagents

    1.   Aqueous trichloroacetic acid (100 g/l).

    2.   Aqueous hydrochloric acid (6 mol/l).

    3.   Aqueous sodium nitrite solution (100 g/l, freshly prepared).

    4.   Aqueous ammonium sulfamate solution (150 g/l).

    5.   Aqueous sodium hydroxide solution (6 mol/l).

    Standards

         Prepare solutions containing paracetamol at concentrations of 0,
    50, 100, 200 and 400 mg/l in blank plasma. These solutions are
    unstable even at 4C and must be prepared weekly or stored at -20C.

    Method

    1.   Add 2 ml of trichloroacetic acid to 1 ml of sample or standard,
         mix and centrifuge for 5 minutes

    2.   In a separate tube add 1 ml of hydrochloric acid to 2 ml of
         sodium nitrite solution and mix.  Take care - brown nitrogen
          dioxide fumes may be evolved.

    3.   Add 2.0 ml of the supernatant from step 1 to the mixture obtained
         in step 2, mix, and allow to stand for 2-3 minutes at room
         temperature.

    4.   Add 2 ml of ammonium sulfamate solution drop by drop to remove
         excess nitrous acid.  Take care - vigorous frothing occurs.

    5.   Add 2 ml of sodium hydroxide solution, vortex-mix to remove any
         gas bubbles and measure the absorbance at 450 nm against a plasma
         blank (see section 4.5.2).

    Results

         Calculate the plasma paracetamol concentration by comparison with
    the results obtained from the standard solutions. Paracetamol
    metabolites do not interfere, but the method is only useful within
    4-24 hours of ingestion and the limit of sensitivity (normally
    50 mg/l) may be 100 mg/l or more with uraemic sera.

         Salicylic acid interferes to a small extent: a salicylate
    concentration of 1 g/l gives an apparent paracetamol concentration of
    50 mg/l. However, 4-aminosalicylic acid reacts strongly (100 mg/l
    gives an apparent paracetamol concentration of 320 mg/l). Levodopa
    also interferes, and specimens contaminated with mucous heparin or
    other solutions containing  o-cresol preservative can give very high
    false readings.

    Sensitivity

         Paracetamol, 50 mg/l.

    Clinical interpretation

         Following paracetamol overdosage only mild symptoms, such as
    nausea and vomiting, may occur initially, but severe, possibly fatal,
    hepatic damage may develop within days of the ingestion. Renal damage
    also occurs in a proportion of patients. Treatment with methionine or
    acetylcysteine ( N-acetylcysteine) can protect against such damage if
    given within 12-15 hours of the overdose (see Table 6).

         Since indicators of hepatic damage such as prothrombin time (see
    section 3.2.1) may only become abnormal at 12-36 hours, measurement of
    the plasma paracetamol concentration is important not only in
    establishing the diagnosis, but also in assessing the need for
    protective therapy (Fig. 12). However, the qualitative urine test
    given above should be performed if there is any suspicion that
    paracetamol has been ingested, especially in patients presenting 24
    hours or more after ingestion.

    FIGURE 12

    6.84  Paraquat

    1,1'-Dimethyl-4,4'-bipyridylium ion; C12H14N2; relative molecular
    mass, 186

    CHEMICAL STRUCTURE 42

         Paraquat is a widely used contact herbicide and may be formulated
    together with the related herbicide diquat. Paraquat is often
    encountered as the dichloride and is extremely poisonous - the lethal
    dose in an adult may be as low as 4 mg/kg of body weight. Paraquat and
    diquat give highly coloured products with sodium dithionite, and this
    reaction forms the basis of the test described.

    Qualitative test

         Applicable to urine, stomach contents and scene residues.

    Reagents

    1.   Sodium dithionite (solid, stored in a desiccator).

    2.   Aqueous ammonium hydroxide (2 mol/l).

    3.   Blank urine.

    4.   Urine specimen containing paraquat ion (10 mg/l).  Take care
          paraquat is very toxic and may be absorbed through the skin.

    Method

    1.   Add 0.5 ml of ammonium hydroxide solution to 1 ml of test
         solution, and to 1-ml portions of blank and standard urines in
         separate test-tubes.

    2.   Add about 20 mg of sodium dithionite to each tube and mix.

    3.   If any colour forms in the test solution, agitate in air for
         several minutes.

    Results

         A blue/blue-black colour indicates the presence of paraquat. The
    related herbicide diquat gives a yellow-green colour, but interference
    from this compound is insignificant in the presence of paraquat.

         If the colour fades on continued agitation in air, paraquat/
    diquat is confirmed - the original colour can be restored by adding
    more sodium dithionite.

    Sensitivity

         Paraquat, 1 mg/l.

    Clinical interpretation

         Ingestion of paraquat may cause a burning sensation in the mouth,
    oesophagus and abdomen together with ulceration of the lips, tongue
    and pharynx. After massive absorption of paraquat, the patient usually
    dies quickly from multiple organ failure. Absorption of lower doses
    may lead to the development of progressive pulmonary fibrosis, which
    ultimately causes death from respiratory failure. Myocardial and renal
    failure may also occur. Treatment is symptomatic and supportive.
    Measures to reduce absorption (by the oral administration of Fuller's
    earth or activated charcoal) or enhance elimination (by haemodialysis)
    of paraquat have not been shown to affect the outcome.

         The major role of an analysis is to assess the prognosis in
    patients at risk from progressive pulmonary fibrosis; a strongly
    positive result in a urine sample obtained more than 4 hours after
    ingestion indicates a poor prognosis. Plasma paraquat concentrations
    can be measured as a prognostic guide, but reliable methods require
    either radioimmunoassay or high-performance liquid chromatography.

    6.85  Pentachlorophenol

    PCP; C6HCl5O; relative molecular mass, 266

    CHEMICAL STRUCTURE 43

         Pentachlorophenol is widely used in wood preservatives and
    disinfectants, and as a contact herbicide. Pentachlorophenol uncouples
    oxidative phosphorylation, and poisoning can occur as a result of
    occupational exposure as well as ingestion. Unlike the dinitrophenol
    pesticides, there is no yellow staining of the skin, but
    pentachlorophenol has a characteristic phenolic smell and this may
    help the diagnosis.

    Qualitative test

         Applicable to stomach contents and scene residues.

    Reagents

    1.   Aqueous sodium hydroxide solution (2 mol/l).

    2.   Concentrated sulfuric acid (relative density 1.83).

    3.   Concentrated nitric acid (relative density 1.40).

    Method

    1.   Mix 10 ml of sample and 20 ml of  n-butyl acetate for 5 minutes
         using a rotary mixer and centrifuge for 5 minutes.

    2.   Transfer the extract to a clean tube and evaporate to dryness on
         a boiling water-bath under a stream of compressed air or
         nitrogen.

    3.   Add 0.2 ml of concentrated nitric acid to the residue and heat
         the tube in the boiling water-bath for 30 seconds.

    4.   Cool and add 0.1 ml of the mixture to 2 ml of concentrated
         sulfuric acid.

    5.   To the remainder of the cooled mixture add 2 ml of purified water
         and then add sodium hydroxide solution drop by drop until the pH
         reaches 8 (universal indicator paper).

    Results

         Pentachlorophenol gives a red colour in steps 3 and 4 and a
    brown-violet colour in step 5. Other chlorinated phenols such as
    hexachlorophane also react in this test.

    Sensitivity

         Pentachlorophenol, 1 g/l.

    Clinical interpretation

         Exposure to pentachlorophenol may cause sweating, hyperpyrexia,
    increased respiratory rate and tachycardia. Death has occurred in
    severe cases. Since acute poisoning occurs commonly by skin and
    pulmonary absorption, the prevention of further absorption is
    important as well as symptomatic and supportive measures.

    6.86  Peroxides

         Hydrogen peroxide (H2O2) is an oxidizing agent used as a bleach
    and sterilizing agent in cosmetics and other household products, and
    in industry. It is often encountered as a relatively dilute aqueous
    solution (60 ml/l or "20 volume", meaning that 1 volume of liquid can
    release 20 volumes of oxygen), but concentrations of up to 300 ml/l
    ("100 volume") are used in industry.

         Solid metallic peroxides such as barium peroxide (BaO2) and
    magnesium peroxide (MgO2) are very strong oxidizing agents. These
    compounds have various industrial uses and liberate hydrogen peroxide
    on treatment with dilute acid. Some organic peroxides are used as
    catalysts in the production of epoxy resins.

    Qualitative test

         Applicable to stomach contents and scene residues.

    Reagents

    1.   Aqueous hydrochloric acid (2 mol/l).

    2.   Aqueous potassium dichromate solution (100 g/l).

    3.   Aqueous sulfuric acid (2 mol/l).

    Method

    1.   If the suspect material is a solid, carefully prepare a paste
         (about 1 g) with water and add to 10 ml of cold dilute
         hydrochloric acid.

    2.   Add 1 ml of liquid test solution (or 1 ml of the acidified
         solution prepared above) to 1 ml of potassium dichromate
         solution, 1 ml of dilute sulfuric acid and 2 ml of diethyl ether.

    3.   Vortex-mix for 30 seconds and allow the phases to separate.

    Results

         A blue colour in the ether layer indicates the presence of
    hydrogen peroxide, either in the test solution or by liberation from a
    metal peroxide.

    Sensitivity

         Hydrogen peroxide, 100 mg/l.

    Confirmatory test

         Applicable to stomach contents and scene residues.

    Reagents

    1.   Aqueous lead acetate solution (100 g/l).

    2.   Hydrogen sulfide gas (cylinder).  Avoid inhalation of hydrogen
          sulfide - it has a strong smell of rotten eggs at low
          concentration and is very toxic.

    Method

    1.   Soak a strip of filter-paper in the lead acetate solution, expose
         to hydrogen sulfide  in a fume cupboard, and allow to dry.

    2.   Spot 0.1 ml of liquid test solution or 0.1 ml of the acidified
         solution prepared for the qualitative test on the paper.

    Results

         A white spot is formed on the brown-black paper if hydrogen
    peroxide is present owing to the oxidation of lead sulfide to sulfate.

    Sensitivity

         Hydrogen peroxide, 500 mg/l.

    Clinical interpretation

         Ingestion of hydrogen peroxide gives rise to a burning sensation
    in the mouth, throat and oesophagus. However, there are usually no
    primary systemic effects, since decomposition to water and oxygen
    occurs before absorption. Poisoning with metallic peroxides is very
    rare, but these compounds are powerful oxidizing and corrosive agents
    and can give rise to systemic toxicity attributable to the metallic
    component. Treatment is symptomatic and supportive.

    6.87  Pethidine

    Meperidine; ethyl 1-methyl-4-phenylpiperidine-4-carboxylate;
    C15H21NO2; relative molecular mass, 247

    CHEMICAL STRUCTURE 44

         Pethidine is a narcotic analgesic. About 45% of an oral dose is
    metabolized by  N-demethylation to norpethidine, by hydrolysis to
    pethidinic acid, and by a number of other pathways. Up to
    approximately 30% of a dose is excreted in urine as pethidine and
    norpethidine under acidic conditions, but only 5% is excreted as these
    compounds if the urine is alkaline.

         There is no simple qualitative test for pethidine, but this
    compound and its metabolites can be detected and identified by thin-
    layer chromatography of a basic solvent extract of urine (see section
    5.2.3).

    Clinical interpretation

         Acute overdosage with pethidine may give rise to pin-point
    pupils, hypotension, hypothermia, coma and convulsions. Death may
    ensue from profound respiratory depression, but fatalities are
    relatively rare. Naloxone rapidly reverses the central toxic effects
    of pethidine (see section 2.2.2).

    6.88  Petroleum distillates

         Petrol (gasoline) is largely a mixture of normal and branched-
    chain aliphatic hydrocarbons (C4-C12) with boiling points in the
    range 39-204C. Paraffin (kerosene) is a higher boiling fraction.
    Acute poisoning with petrol usually arises from inhalation of vapour
    as a result of industrial accident or deliberate abuse (sniffing or
    ingestion). Tetraethyl lead is frequently added to petrol to prevent
    pre-ignition (anti-knock) and chronic organo-lead poisoning can follow
    long-term abuse.

         There are no simple qualitative tests for petroleum distillates.
    However, the characteristic smell of petrol on the breath or from
    stomach contents (or even postmortem tissue) may help to indicate the
    diagnosis.

    Clinical interpretation

    Headache, dizziness, nausea, vomiting, confusion, tremor,
    disorientation, coma and cardiac arrhythmias may occur after oral
    ingestion of petrol or sublethal inhalation of petrol vapour. The
    aspiration of even small quantities of petrol may lead to chemical
    pneumonitis. The inhalation of high concentrations of vapour may be
    rapidly fatal, with acute respiratory failure or cardiorespiratory
    arrest.

    6.89  Phenacetin

     p-Ethoxyacetanilide; acetophenetidin; C10H13NO2; relative
    molecular mass, 179

    CHEMICAL STRUCTURE 45

         Phenacetin was previously used as an analgesic, but long-term use
    was associated with nephrotoxicity. It is largely metabolized to
    paracetamol and thus ingestion of phenacetin can be detected in urine
    using the  o-cresol/ammonia test on a hydrolysed urine specimen.

    Qualitative test

         Applicable to urine,  o-Cresol/ammonia test - see paracetamol
    monograph (section 6.83).

    Results

         A strong, royal blue colour developing immediately indicates the
    presence of paracetamol. This test is very sensitive and will detect
    therapeutic dosage with phenacetin 24-48 hours later.

         Only aromatic amines such as aniline, which also give rise to
     p-aminophenol in urine after hydrolysis, are known to interfere.
    Ethylenediamine (from aminophylline, for example; see section 6.105)
    gives a green colour in this test.

    Sensitivity

          p-Aminophenol, 1 mg/l.

    Clinical interpretation

         Acute ingestion of phenacetin can cause dizziness, euphoria,
    cyanosis, haemolytic anaemia, respiratory depression, and
    cardiorespiratory arrest. Methaemoglobinaemia is often produced and
    may be indicated by dark chocolate-coloured blood (see section 3.2.2).
    Blood methaemoglobin can be measured, but is unstable and the use of
    stored samples is unreliable. Although metabolized to paracetamol,
    phenacetin does not cause acute hepatorenal necrosis. Treatment is
    symptomatic and supportive.

    6.90  Phenols

         Phenol (hydroxybenzene; carbolic acid; C6H5OH; relative
    molecular mass, 94) and cresol (cresylic acid; CH3.C6H4OH; relative
    molecular mass, 108) are used as disinfectants and in the plastics
    industry. Commercial cresol is a mixture of  o-, m-, and  p-cresols
    in which the  m-isomer predominates. The estimated minimum lethal
    dose of phenol or cresol in an adult is 1-2 g.

         Both phenol and cresol are readily absorbed through the skin and
    via the gastrointestinal tract when ingested. They are excreted in
    urine mainly as glucuronide or sulfate conjugates.

    Qualitative test

         Applicable to urine.

    Reagents

    1.   Folin-Ciocalteau reagent. Dissolve 100 g of sodium tungstate and
         25 g of sodium molybdate in 800 ml of purified water in a
         1.5-l flask. Add 50 ml of concentrated orthophosphoric acid
         (840-900 g/kg) and 100 ml of concentrated hydrochloric acid
         (relative density 1.18) and reflux for 10 hours. Cool, add 150 g
         of lithium sulfate, 50 ml of purified water and 0.5 ml of
         elemental bromine, and allow to stand for 2 hours. Boil for 15
         minutes to remove excess bromine, cool, filter if necessary, and
         dilute to 1 litre with purified water. This solution is yellow
         and should be stable for 4 months if stored at 4C. Folin-
         Ciocalteau reagent can also be purchased ready-made.

    2.   Aqueous sodium hydroxide solution (2 mol/l).

    Method

    1.   Dilute 1 ml of Folin-Ciocalteau reagent with 2 ml of purified
         water and add 1 ml of urine.

    2.   Add 1 ml of sodium hydroxide solution and vortex-mix for 5
         seconds.

    Results

         A blue colour indicates the presence of a phenolic compound.
    Halogenated phenols such as 2,4,6-trichlorophenol react less strongly
    than nonhalogenated phenols.

    Sensitivity

         Phenol, 10 mg/l.

    Clinical interpretation

         Phenols burn and cause depigmentation of the skin, and corrode
    the lips and mouth if ingested. In severe poisoning, nausea, vomiting,
    abdominal pain, gastric haemorrhage or perforation, metabolic
    acidosis, coma, hypotension and shock may occur. Death from
    respiratory depression may ensue. Hepatorenal failure is an additional
    complication, and the urine may become dark-coloured owing to the
    presence of free haemoglobin. Apart from skin decontamination with
    castor oil or olive oil, treatment is symptomatic and supportive.

    6.91  Phenothiazines

         These compounds are derivatives of phenothiazine, which itself is
    used as an anthelminthic in veterinary medicine.

    CHEMICAL STRUCTURE 46

         Phenothiazines are widely used as antihistamines, tranquillizers,
    and in various psychiatric disorders. They are often extensively
    metabolized. Chloropromazine, for example, has over 50 metabolites in
    humans. The test described below is based on the reaction of many of
    these compounds with ferric ion under acidic conditions.

         Phenothiazines are often detected during thin-layer
    chromatography of basic solvent extracts of urine (see section 5.2.3),
    but specific identification of the compound ingested may be impossible
    if only urine is available. Low-dose phenothiazines, such as
    fluphenazine, may not be detectable in urine using either method.

    Some commonly encountered phenothiazines are listed in Table 33.

    Table 33.  Some common phenothiazines
                                                                        

    Compound           Chemical name                           Relative
                                                               molecular
                                                                 mass
                                                                        

    Chlorpromazine     3-(2-Chlorophenothiazin-10-yl)-           319
                       N,N-dimethylpropylamine
    Chlorprothixene    (Z)-3-(2-Chlorothioxanthen-9-ylidene)-    316
                       N,N-dimethylpropylamine
    Dimetotiazine      10-(2-Dimethylaminopropyl)-N,N-           392
                       dimethylphenothiazine-2-sulfonamide
    Prochlorperazine   2-Chloro-10-[3-(4-methylpiperazin-1-yl)   374
                       propyl]phenothiazine
    Promazine          N,N-Dimethyl-3-(phenothiazin-10-yl)       284
                       propylamine
    Promethazine       1,N,N-Trimethyl-2-(phenothiazin-10-yl)    284
                       ethylamine
    Thioridazine       10-[2-(1-Methyl-2-piperidyl)ethyl]-2-     371
                       methylthiophenothiazine
                                                                        

    Qualitative test

         Applicable to urine, stomach contents and scene residues.

    Reagent

         FPN reagent. Mix 5 ml of aqueous ferric chloride solution
    (50 g/l), 45 ml of aqueous perchloric acid (200 g/kg) and 50 ml of
    aqueous nitric acid (500 ml/l).

    Method

         Add 1 ml of FPN reagent to 1 ml of sample and mix for 5 seconds.

    Results

         Colours ranging from pink, red or orange to violet or blue may
    indicate the presence of phenothiazines or metabolites. Urine from
    patients on chronic treatment with conventional phenothiazines, such
    as chlorpromazine, will usually give a positive reaction.

         Tricyclic antidepressants such as imipramine may also give green
    or blue colours. False positive reactions may be obtained in patients
    with phenylketonuria or hepatic impairment.

    Sensitivity

         Chlorpromazine, 25 mg/l.

    Clinical interpretation

         Features of acute poisoning with phenothiazines include
    drowsiness, tremor, restlessness, hyperreflexia, hypothermia,
    hypotension, hypoventilation, convulsions, tachycardia and cardiac
    arrhythmias. Phenothiazine overdosage is not normally associated with
    a fatal outcome, although serious poisoning with, for example,
    chlorpromazine has been described. Treatment is normally symptomatic
    and supportive.

    6.92  Phenytoin

    Diphenylhydantoin; 5,5-diphenylimidazolidine-2,4-dione; C15H12N2O2;
    relative molecular mass, 252

    CHEMICAL STRUCTURE 47

         Phenytoin is a widely used anticonvulsant. Metabolic pathways
    include aromatic hydroxylation and conjugation, less than 5% of a dose
    being excreted unchanged in urine. The estimated minimum lethal dose
    in an adult is 5 g, but few fatal overdoses involving this compound
    alone have been reported.

         There is no simple qualitative test for phenytoin, but it can be
    detected and identified by thin-layer chromatography of an acidic
    solvent extract of urine, stomach contents or scene residues (see
    section 5.2.3).

    Clinical interpretation

         Features of phenytoin poisoning include tremor, nystagmus,
    ataxia, coma and respiratory depression. Intravenous overdosage may be
    associated with cardiac toxicity. Treatment is normally symptomatic
    and supportive. Haemoperfusion may be of value in severe cases.

    6.93  Phosphorus and phosphides

         Yellow phosphorus (P) and the phosphides of zinc, aluminium and
    magnesium are used as rodenticides, usually as a paste containing
    sugar and bran.

    Qualitative test

         Applicable to stomach contents and scene residues.

    Reagents

    1.   Silver nitrate solution (saturated) in methanol.

    2.   Aqueous lead acetate solution (100 g/l).

    Method

    1.   Soak a strip (5  1 cm) of filter-paper in the silver nitrate
         solution and allow to dry at room temperature.

    2.   Soak a similar strip of filter-paper in the lead acetate solution
         and again dry at room temperature.

    3.   Place 5 ml of sample in a boiling-tube fitted with a cork with a
         slit cut in each side.

    4.   Insert the test papers into the slits, stopper the tube and heat
         on a water-bath at 60C for 20 minutes.

    Results

         If only the silver nitrate paper is blackened then phosphorus or
    phosphides may be present. If both papers are blackened then sulfides
    may be present and the result is inconclusive.

    Sensitivity

         Phosphorus, 1 g/l.

    Confirmatory test

         Applicable to blackened silver nitrate paper from the test above.

    Reagents

    1.   Ammonium molybdate reagent. Mix 5 g of ammonium molybdate in
         100 ml of water and 35 ml of concentrated nitric acid (relative
         density 1.42).

    2.    o-Toluidine reagent. Mix 50 mg of  o-toluidine and 10 ml of
         glacial acetic acid, diluted to 100 ml with purified water.

    3.   Concentrated ammonium hydroxide (relative density 0.88).

    4.   Powdered calcium hypochlorite.

    Method

    1.   Place the silver nitrate paper on a glass microscope slide and
         cover with calcium hypochlorite.

    2.   Leave in a moist chamber for 15 minutes to allow oxidation of
         phosphide to phosphate.

    3.   Remove excess hypochlorite by careful washing with a small amount
         of purified water and dry the test paper by blotting with
         absorbent tissue.

    4.   Add 50 l of ammonium molybdate reagent to the dried paper
         followed by 50 l of  o-toluidine reagent and expose the paper
         to ammonia fumes from concentrated ammonium hydroxide  in a
          fume cupboard.

    Results

         A blue colour confirms phosphorus.

    Sensitivity

         Phosphorus, 1 g/l.

    Clinical interpretation

         Acute poisoning with yellow phosphorus gives rise to
    gastrointestinal corrosion, nausea and vomiting, leading to coma,
    hypotension and hepatorenal damage. Phosphides release phosphine
    (PH3) on contact with water or moist air, and this gas acts on the

    gastrointestinal and central nervous systems. Abdominal pain may be
    followed by nausea, vomiting, gross ataxia, convulsions and coma, with
    death, usually within 2 hours, in severe cases. Treatment is
    symptomatic and supportive.

    6.94  Procainamide

    4-Amino- N-(2-diethylaminoethyl)benzamide;C13H21N3O; relative
    molecular mass, 235

    CHEMICAL STRUCTURE 48

         Procainamide is a widely used antiarrhythmic drug. The major
    metabolite,  N-acetylprocainamide (NAPA) has similar pharmacological
    activity to the parent compound. Up to 80% of a dose is excreted in
    urine in 24 hours, some 50-60% as procainamide and about 30% as NAPA.
    The normal oral dose is 0.5-1 g of procainamide every 4-6 hours, but
    as little as 200 mg given intravenously may prove fatal.

         There is no simple qualitative test for procainamide, but this
    compound and NAPA can be detected and identified by thin-layer
    chromatography of a basic solvent extract of urine (see section
    5.2.3).

    Clinical interpretation

         Acute poisoning with procainamide may cause anorexia, nausea,
    vomiting, diarrhoea and cardiac arrhythmias. Rapid intravenous
    injection may cause hypotension, convulsions and collapse. Treatment
    is symptomatic and supportive.

    6.95  Propan-2-ol

     iso-Propanol;  iso-propyl alcohol; CH3.CHOH.CH3; relative
    molecular mass, 60

         Propan-2-ol is used in lotions for topical administration, in
    window and screen washers, and as a solvent for toiletries. It is also
    used as a vehicle for certain pharmaceutical preparations and serious
    iatrogenic poisoning has occurred in children. The estimated minimum
    fatal dose of propan-2-ol in an adult is 240 ml.

         Propan-2-ol is metabolized to acetone by alcohol dehydrogenase.
    The qualitative test described below relies on the oxidation of
    propan-2-ol to acetone and subsequent detection of acetone. Note that
    propan-2-ol is often used as a topical antiseptic prior to
    venepuncture, and care must be taken to avoid contamination of the
    sample if poisoning with this agent is suspected.

    Qualitative test

         Applicable to plasma or serum.

    Reagents

    1.   Salicylaldehyde solution (100 ml/l) in methanol.

    2.   Aqueous sodium hydroxide solution (300 g/l).

    3.   Potassium permanganate reagent. Mix 3 g of potassium
         permanganate, 15 ml of orthophosphoric acid (850 g/kg) and 85 ml
         of purified water.

    4.   Aqueous trichloroacetic acid solution (200 g/l).

    5.   Sodium bisulfite (solid).

    Method

    1.   Add 1 ml of plasma or serum to 2 ml of trichloroacetic acid
         solution, vortex-mix for 30 seconds and centrifuge for 5 minutes.

    2.   Transfer 1 ml of the supernatant to a second tube and add 0.3 ml
         of potassium permanganate reagent.

    3.   Vortex-mix for 5 seconds and allow to stand for 10 minutes. If
         the pink coloration fades continue to add 0.1-ml portions of
         potassium permanganate reagent until the colour persists.

    4.   Decolorize by adding solid sodium bisulfite (about 100 mg).

    5.   Add 3 ml of sodium hydroxide solution and 0.1 ml of
         salicylaldehyde solution and vortex-mix for 5 seconds.

    6.   Heat in a boiling water-bath for 4 minutes and cool.

    Results

         A red colour indicates the presence of propan-2-ol or acetone.

    Sensitivity

         Propan-2-ol, 50 mg/l.

    Clinical interpretation

         Initial symptoms of poisoning with propan-2-ol are similar to
    those of ethanol and include inebriation, nausea, vomiting and
    abdominal pain. Serious poisoning is rare but features include
    haemorrhagic gastritis, coma, respiratory depression, hypothermia,
    renal failure, rhabdomyolysis, myoglobinuria, haemolytic anaemia and
    ketonuria. Treatment is supportive and may include haemodialysis in
    severe cases.

         The half-life of acetone is much longer than that of propan-2-ol
    so that the later signs of poisoning observed are largely those of
    acetone. Acetone can be detected on the breath of patients poisoned
    with propan-2-ol. A simple dip-strip test for urinary acetone is
    available.

    6.96  Propranolol

    ()-1-Isopropylamino-3-(1-naphthyloxy)propan-2-ol; C16H21NO2;
    relative molecular mass, 259

    CHEMICAL STRUCTURE 49

         Propranolol is a -adrenoceptor-blocking agent (-blocker). It is
    given orally in the treatment of hypertension and some cardiac
    disorders, and has a variety of other uses. Propranolol undergoes
    extensive first-pass metabolism, and metabolic pathways include
    aromatic hydroxylation,  N-dealkylation, oxidative deamination and
    conjugation.

         There is no simple qualitative test for propranolol, but this
    compound and some of its metabolites may be detected and identified by
    thin-layer chromatography of a basic solvent extract of urine (see
    section 5.2.3).

    Clinical interpretation

         Overdosage with propranolol and other -blockers may cause
    delirium, hallucinations, bradycardia, hypotension, bronchospasm,
    hypoglycaemia, coma and convulsions. Death may follow low-output
    cardiac failure or cardiorespiratory arrest. Treatment may include the
    administration of atropine, glucagon and -agonists.

    6.97  Propylene glycol

    Propane-1,2-diol; CH3.CHOH.CH2OH; relative molecular mass, 76

         Propylene glycol is widely used as a solvent in the
    pharmaceutical and food industries, usually as a 100 ml/l or 200 ml/l
    aqueous solution, and in veterinary practice. It has a relatively
    short plasma half-life (4-8 hours), and is metabolized primarily to
    lactic and pyruvic acids, although a large proportion (20-30%) of a
    dose is excreted unchanged in urine.

         Propylene glycol is relatively safe in normal use, but cases of
    poisoning associated with its use as a vehicle for drugs or vitamins
    given intravenously or orally have been described. Plasma
    concentrations associated with serious toxicity are over 4 g/l,
    corresponding in adults to the administration of 100-200 ml of
    intravenous fluids containing 200-300 ml/l propylene glycol over a
    relatively short time.

         There is no simple qualitative test for propylene glycol.

    Clinical interpretation

         Overdosage with propylene glycol may cause lactic acidosis,
    haemolysis, coma, convulsions and cardiorespiratory arrest. Increased
    plasma osmolality can be a useful but nonspecific indicator of
    poisoning with this compound (see section 3.1.3).

    6.98  Quinine and quinidine

         These compounds have the structure:

    CHEMICAL STRUCTURE 50

    Quinine ((8S, 9R)-6'-methoxycinchonan-9-ol trihydrate;
    C20H24N2O23H2O; relative molecular mass, 379) is
    the dextrorotatory stereoisomer of quinidine ((8r, 9s)-6'-
    methoxycinchonan-9-ol dihydrate; C20H24N2O22H2O; relative
    molecular mass, 361). Commercial samples of either compound may
    contain up to 10% or 30% of hydroquinine or hydroquinidine,
    respectively.

         Quinine is the major alkaloid found in the bark of various
    species of  Cinchona and is used in the treatment of malaria. It is
    also used to treat night cramps and is a constituent of tonic water.
    The fatal dose of quinine in an adult may be as little as 8 g.
    Quinidine is used as an antiarrhythmic. Both compounds are extensively
    metabolized, largely to hydroxylated metabolites.

    Qualitative test

         Applicable to urine.

    Reagents

    1.   Aqueous hydrochloric acid (2 mol/l).

    2.   Sodium chloride (solid).

    Method

    1.   Add 0.1 ml of dilute hydrochloric acid to 1 ml of sample and
         vortex-mix for 10 seconds.

    2.   Examine under ultraviolet light (366 nm).

    3.   If fluorescence is observed, add about 1 g of sodium chloride and
         vortex-mix for 30 seconds.

    Results

         If any fluorescence observed at step 2 is due to quinine/
    quinidine then it will be largely, if not completely, quenched by
    addition of sodium chloride.

         In addition to the above, quinine and quinidine and their
    metabolites can be detected and identified by thin-layer
    chromatography of a basic solvent extract of urine (see section
    5.2.3). However, care may be needed to differentiate these compounds
    from emetine if syrup of ipecacuanha has been used to induce vomiting
    (see section 2.2.1 and Table 15).

    Sensitivity

         Quinine or quinidine, 50 mg/l.

    Clinical interpretation

         Overdosage with quinine may cause nausea, vomiting, abdominal
    pain, diarrhoea, tinnitus, deafness, vertigo, headache, blurred
    vision, blindness (which may be permanent), hypotension, coma, acute
    renal failure and cardiorespiratory arrest. In addition to general
    supportive measures, repeat-dose oral activated charcoal may be used
    to enhance systemic elimination of quinine. The efficacy of stellate
    ganglion block in preventing retinal damage has not been established.

         The gastrointestinal and cerebellar signs of acute quinidine
    poisoning are similar to those caused by quinine. However, metabolic
    and circulatory effects predominate, and include hypotension,
    hypokalaemia, hypocalcaemia, hypophosphataemia, hypomagnesaemia,
    metabolic acidosis, acute renal failure, coma, convulsions, cardiac
    arrhythmias and circulatory collapse. Treatment is largely symptomatic
    and supportive.

    6.99  Salicylic acid and derivatives

    Salicylic acid

    2-Hydroxybenzoic acid; C7H6O3; relative molecular mass, 138

    CHEMICAL STRUCTURE 51

         Salicylic acid is used topically to treat various dermatological
    disorders. It is the principal plasma metabolite of acetylsalicylic
    acid and can also arise from the metabolism of methyl salicylate and
    salicylamide. Salicylic acid is excreted in the urine, mostly as a
    conjugate with glycine (salicyluric acid).

         The salicylic acid derivatives described below are commonly
    encountered drugs.

    Acetylsalicylic acid

    Aspirin; C9H8O4; relative molecular mass, 180

    CHEMICAL STRUCTURE 52

         Acetylsalicylic acid is the most frequently used salicylic acid
    derivative. It is used as an analgesic and is also a metabolite of
    aloxiprin and benorilate. The estimated minimum lethal dose in an
    adult is 15 g.

         Acetylsalicylic acid is rapidly metabolized by plasma esterases
     in vivo to salicylic acid, which is then excreted in the urine,
    mostly as a conjugate with glycine (salicyluric acid).

    4-Aminosalicylic acid

     p-Aminosalicylic acid; PAS; 4-amino-2-hydroxybenzoic acid;
    C7H7NO3; relative molecular mass, 151

    CHEMICAL STRUCTURE 53

         4-aminosalicylic acid is used in the treatment of tuberculosis.

    Methyl salicylate

    Methyl 2-hydroxybenzoate; salicylic acid methyl ester; C8H8O3;
    relative molecular mass, 152

    CHEMICAL STRUCTURE 54

         Methyl salicylate (oil of wintergreen) is a strong-smelling
    liquid at room temperature and is widely used in topical medicinal
    products. On ingestion it is more toxic than acetylsalicylic acid
    because it is more rapidly absorbed. Deaths have occurred in children
    after ingestion of as little as 4 ml; 30 ml is usually fatal in
    adults.

         Methyl salicylate is partially metabolized to salicylic acid
     in vivo.

    Salicylamide

    2-Hydroxybenzamide; C7H7NO2; relative molecular mass, 137

    CHEMICAL STRUCTURE 55

         Salicylamide is used as an analgesic. On hydrolysis, it forms
    salicylic acid.

         Salicylates give a distinctive purple colour with ferric ions
    and this reaction forms the basis of the test described. A simple
    dip-strip test for salicylates based on this reaction is available.

         Acetylsalicylic acid and methyl salicylate do not themselves
    react with ferric ions, so that stomach contents and scene residues
    must be hydrolysed before analysis is performed. Salicylamide is only
    detectable after hydrolysis, even in urine samples.

    Qualitative test

         Applicable to urine, stomach contents and scene residues.

    Reagent

         Trinder's reagent. Mix 40 g of mercuric chloride dissolved in
    850 ml of purified water with 120 ml of aqueous hydrochloric acid
    (1 mol/l) and 40 g of hydrated ferric nitrate, and dilute to 1 litre
    with purified water.

    Method

         Add 0.1 ml of Trinder's reagent to 2 ml of sample and mix for 5
    seconds.

         To test for acetylsalicylic acid or methyl salicylate in stomach
    contents or scene residues, and to test for salicylamide in urine,
    stomach contents or scene residues, first boil 1 ml of sample with
    1 ml of aqueous hydrochloric acid (0.1 mol/l) for 10 minutes, cool,
    filter if necessary, and then neutralize with 1 ml of aqueous sodium
    hydroxide (0.1 mol/l).

    Results

         A strong violet colour indicates the presence of salicylates.
    Azide preservatives react strongly in this test, and weak false
    positives can be given by urine specimens containing high
    concentrations of ketones (ketone bodies).

         This test is sensitive and will detect therapeutic dosage with
    salicylic acid, acetylsalicylic acid, 4-aminosalicylic acid, methyl
    salicylate and salicylamide.

    Sensitivity

         Salicylate, 10 mg/l.

    Quantitative assay

         Applicable to plasma or serum (1 ml).

    Reagent

         Trinder's reagent (see above).

    Standards

         Aqueous solutions containing salicylic acid at concentrations of
    0, 200, 400 and 800 mg/l. Store at 4C when not in use.

    Method

    1.   Add 5 ml of Trinder's reagent to 1 ml of sample or standard.

    2.   Vortex-mix for 30 seconds and centrifuge for 5 minutes.

    3.   Measure the absorbance of the supernatant at 540 nm against a
         plasma blank (see section 4.5.2).

    Results

         Calculate the plasma salicylate concentration from the graph
    obtained on analysis of the salicylate standards. Some salicylate
    metabolites interfere, but plasma concentrations of these compounds
    are usually low. Oxalates, for example, from fluoride/oxalate blood
    tubes, also interfere in this test.

    Sensitivity

         Salicylate, 50 mg/l.

    Clinical interpretation

         The topical use of salicylic acid and methyl salicylate
    and ingestion of salicylates may give rise to features of
    salicylism. Respiratory alkalosis followed by metabolic acidosis is
    characteristic, although in practice a mixed acid-base disturbance is
    usually seen. The results of blood gas analyses are an important guide
    to the severity of poisoning (see section 3.1.2). If acute poisoning
    is suspected, the plasma salicylate concentration should be measured
    using the method described above.

         Active measures to correct acid-base status and urinary
    alkalinization to enhance elimination of the poison may be considered,
    depending on the patient's condition and the plasma salicylate
    concentration. Repeated oral activated charcoal may also be employed
    (see section 2.2.3).

         Serial plasma salicylate and urine pH measurements are valuable
    in monitoring active treatment. A guide to the interpretation of
    plasma salicylate results is given in Fig. 13. Concentrations of up to
    300 mg/l may be encountered during therapy in adults.

    FIGURE 13

    6.100  Strychnine

    Strychnidin-10-one; C21H22N2O2; relative molecular mass, 334

    CHEMICAL STRUCTURE 56

         Strychnine and the related compound brucine (10, 11-
    dimethoxystrychnine) are highly toxic alkaloids derived from the seeds
    of  Strychnos nuxvomica and other  Strychnos species. Strychnine has
    a bitter taste and is sometimes used in tonics for this reason. It is
    also used to exterminate rodents and other mammalian pests, and has
    been used to adulterate diamorphine (see section 6.73).

         In addition to the simple tests given below, strychnine can be
    detected and differentiated from brucine by thin-layer chromatography
    of a basic or neutral extract of urine (see section 5.2.3).

    Qualitative test

         Applicable to stomach contents and scene residues.

    Reagents

    1.   Concentrated ammonium hydroxide (relative density 0.88).

    2.   Ammonium vanadate (5 g/l) in concentrated sulfuric acid (relative
         density 1.83).

    Method

    1.   Add 5 ml of sample to 1 ml of concentrated ammonium hydroxide and
         extract with 20 ml of chloroform for 10 minutes using a rotary
         mixer.

    2.   Centrifuge for 10 minutes, remove the upper, aqueous layer and
         transfer the solvent extract to a clean tube.

    3.   Evaporate the extract to dryness under a stream of compressed air
         or nitrogen and dissolve the residue in 100 l of chloroform.

    4.   Transfer 50 l of the reconstituted extract to a porcelain
         spotting tile and add 50 l of ammonium vanadate solution.

    Results

         A violet colour which changes to red and then to yellow over 10
    minutes suggests the presence of strychnine.

    Sensitivity

         Strychnine, 100 mg/l.

    Confirmatory test

         Applicable to stomach contents and scene residues.

    Reagents

    1.   Granulated zinc.

    2.   Concentrated hydrochloric acid (relative density 1.18).

    3.   Aqueous sodium nitrite solution (100 g/l, freshly prepared).

    Method

    1.   Add a granule of zinc to 1 ml of sample and 1 ml of concentrated
         hydrochloric acid and heat in a boiling water-bath for 10
         minutes.

    2.   Cool, remove any remaining zinc and add 50 l of sodium nitrite
         solution.

    Results

         Strychnine gives a pink colour.

    Sensitivity

         Strychnine, 10 mg/l.

    Clinical interpretation

         Ingestion of strychnine can cause convulsions and, notably,
    opisthotonos. Treatment is symptomatic and the patient may need
    intensive supportive care.

    6.101  Sulfides

         Sulfides such as sodium sulfide (Na2S) and calcium sulfide (CaS)
    are used in depilatory agents, luminous paints, ore dressing and
    flotation, dye and plastics manufacture, photography, printing,
    veterinary medicine and a variety of other applications. Ingested
    elemental sulfur is metabolized to sulfide in the gastrointestinal
    tract; the ingestion of 10-20 g of sulfur may cause gastrointestinal
    symptoms. Hydrogen sulfide is often released when metallic sulfides
    are treated with water or acid, and their mammalian toxicity may be
    related to production of this compound.

         Hydrogen sulfide (H2S) is a colourless, extremely toxic gas
    which has, at low concentrations, an unpleasant odour of rotten eggs.
    At higher concentrations the olfactory response is lost and acute
    hydrogen sulfide poisoning is a leading cause of sudden death in the
    workplace. Hydrogen sulfide is released by decomposition of organic
    sulfur-containing materials and from sources of volcanic activity, and
    is used in the plastics, tanning, dye, rubber and petroleum
    industries, among others.

         Hydrogen sulfide is rapidly metabolized  in vivo by oxidation to
    sulfate and other pathways. Any analysis for sulfide in biological
    materials should be performed as quickly as possible, since the
    sulfide ion is unstable in such samples.

    Qualitative test

         Applicable to stomach contents and scene residues.

    Reagents

    1.   Aqueous sulfuric acid (100 ml/l).

    2.   Lead acetate reagent. Mix 50 ml of lead acetate solution (100 g/l
         in boiled, purified water) and 5 ml of aqueous acetic acid
         (2 mol/l).

    Method

    1.   Soak a strip of filter-paper in lead acetate reagent and allow to
         dry.

    2.   Add 3 ml of dilute sulfuric acid to 1 ml of sample, suspend the
         lead acetate-impregnated paper in the neck of the tube and place
         in a boiling water-bath  in a fume cupboard.

    Results

         Sulfides give rise to hydrogen sulfide gas which blackens lead
    acetate paper.

    Sensitivity

         Sulfide, 50 mg/l.

         There is no simple confirmatory test for sulfides; microdiffusion
    methods are unreliable.

    Clinical interpretation

         Exposure to hydrogen sulfide can give rise to headache,
    dizziness, drowsiness, nausea, sore throat, coma, convulsions, cardiac
    arrhythmias, respiratory depression and pulmonary oedema. Treatment
    may include the administration of 100% oxygen and nitrites.

    6.102  Sulfites

         Sulfites such as sodium sulfite (Na2SO3), sodium bisulfite
    (NaHSO3), and sodium metabisulfite (Na2S2O3) are used in the
    paper, water treatment, photographic and textile industries and as
    preservatives in beverages, foods and medications. Sulfur dioxide gas
    (SO2) may be liberated on contact with acid. The estimated fatal dose
    of sodium sulfite in an adult is 10 g.

    Quantitative assay

         Applicable to urine.

    Reagents

    1.   Magenta reagent. Add 0.02 g of basic magenta (fuchsine, CI 42510)
         to 100 ml of aqueous hydrochloric acid (1 mol/l) and store in the
         dark.

    2.   Formaldehyde solution. Dilute 1 ml of methanol-free aqueous
         formaldehyde solution (340-380 g/kg) to 1 litre with purified
         water.

    Standards

         Anhydrous sodium sulfite (1.575 g) in 1 litre of purified water
    (to give a sulfite ion concentration of 1 g/l), diluted with purified
    water to give standards containing sulfite ion at concentrations of
    0.5, 1.5, 3.0, 6.0 and 10.0 mg/l.

    Method

    1.   Add 1 ml of magenta reagent and 3 ml of formaldehyde solution to
         50 l of sample.

    2.   Add 1 ml of magenta reagent and 3 ml of purified water to a
         second 50-l portion of the sample.

    3.   Vortex-mix for 5 seconds and allow to stand for 5 minutes.

    4.   Measure the absorbance of the test solution from step 1 at 570 nm
         using the sample blank from step 2 as reference (see section
         4.5.2).

    Results

         Construct a calibration graph following analysis of the standard
    sulfite solutions and calculate the sulfite concentration in the
    sample.

    Sensitivity

         Sulfite, 0.5 mg/l.

    Clinical interpretation

         Acute sulfite poisoning can cause generalized flush, faintness,
    syncope, wheezing, shortness of breath, cyanosis and cold skin.
    Urticaria and angioedema may be seen within minutes of exposure, and
    may be followed by acute bronchospasm and respiratory arrest in
    certain people. Treatment is symptomatic and supportive. The normal
    urinary concentration of sulfite ion is less than 6 mg/l.

    6.103  Tetrachloroethylene

    Perchlorethylene; tetrachloroethene; CCl2:CCl2; relative molecular
    mass, 166

         Tetrachloroethylene is used as an anthelminthic and as a solvent
    in dry-cleaning and vapour degreasing. Acute poisoning with this
    compound is normally from massive accidental exposure or deliberate
    inhalation (solvent abuse). Only about 0.5% of an absorbed dose of
    tetrachloroethylene is metabolized to trichloroacetic acid, but this
    can be detected in urine using the Fujiwara test.

    Qualitative test

         Applicable to urine. Fujiwara test - see carbon tetrachloride
    monograph (section 6.23).

    Results

         An intense red/purple colour in the upper, pyridine layer
    indicates the presence of trichloro compounds. The blank analysis
    excludes contamination with chloroform from the laboratory atmosphere.

         This test will detect ingestion or exposure to low doses of
    compounds that are extensively metabolized to trichloroacetic acid,
    such as chloral hydrate, dichloralphenazone and trichloroethylene, 12-
    24 hours later. With tetrachloroethylene, the test is correspondingly
    less sensitive, since such a small proportion of a dose is
    metabolized.

    Sensitivity

         Trichloroacetate, 1 mg/l.

    Clinical interpretation

         Signs of poisoning with tetrachloroethylene include ataxia,
    nausea, vomiting, coma, respiratory depression and cardiac
    arrhythmias. Hepatorenal damage is very uncommon. Treatment is
    symptomatic and supportive.

    6.104  Thallium

         Thallium (Tl) salts are employed in the manufacture of
    semiconductors, pigments and lenses, and are used as rodenticides in
    many countries. The lethal dose of thallium in an adult is 0.2-1 g.

         The test given below can be used to give an approximate measure
    of urinary thallium concentration if poisoning with this element is
    suspected.

    Quantitative assay

         Applicable to urine.

    Reagents

    1.   Cyanide reagent. Dissolve 1.6 g of sodium hydroxide, 1.2 g of
         potassium sodium tartrate and 1.36 g of potassium cyanide in
         10 ml of water.  Take care when using concentrated cyanide
          solutions.

    2.   Dithizone solution (250 mg/l) in chloroform (freshly prepared).

    Standards

         Blank urine to which has been added a solution of thallium
    acetate in purified water (1.0-g/l) to give thallium ion
    concentrations of 0.1, 1.0, 5.0 and 10.0 mg/l.

    Method

    1.   Add 1 ml of cyanide reagent to 5 ml of sample or standard in a
         10-ml glass-stoppered test-tube and vortex-mix for 10 seconds.

    2.   Add 2 ml of dithizone solution, vortex-mix for 1 minute, and
         centrifuge for 5 minutes.

    3.   Discard the upper, aqueous layer and filter the chloroform
         extract through phase-separating filter-paper into a clean tube.

    4.   Measure the absorbance of the extract at 480 nm against a blank
         urine extract (see section 4.5.2).

    Results

         A pink/red colour in the lower chloroform layer indicates the
    presence of thallium at concentrations of 1 mg/l or more. Construct a
    calibration graph of absorbance against thallium concentration in the
    standards and measure the thallium concentration in the sample. A
    number of metal ions may interfere in this assay. Atomic absorption
    spectrophotometry is needed in order to measure thallium definitively.

    Sensitivity

         Thallium, 0.1 mg/l.

    Clinical interpretation

         Acute poisoning with thallium salts may lead to gastrointestinal
    stasis, while intermediate and late effects may include disturbances
    of the peripheral and central nervous systems, the cardiorespiratory
    system, eyes and skin. Scalp and facial hair loss is a typical sign of
    thallium poisoning. Patients with urinary thallium concentrations
    exceeding 0.5 mg/l should be treated with potassium
    ferrohexacyanoferrate (Prussian blue; see Table 6) until urinary
    thallium excretion is below 0.5 mg/day.

    6.105  Theophylline

    1,3-Dimethylxanthine; C7H8N4O2; relative molecular mass, 180

    CHEMICAL STRUCTURE 57

         Theophylline is a bronchodilator widely used in the treatment of
    asthma, often as a mixture with ethylenediamine (aminophylline).
    Theophylline is metabolized to 3-methylxanthine, 1,3-dimethyluric
    acid, and 1-methuric acid; caffeine is a metabolite in neonates.

         There is no simple test for theophylline applicable to biological
    fluids. Ethylenediamine gives a green colour in the  o-cresol/ammonia
    urine test used to detect paracetamol, but in this case only indicates
    prior ingestion of aminophylline (see section 6.83).

    Quantitative assay

         Applicable to plasma or serum.

    Reagents

    1.   Tris buffer (0.2 mol/l, pH 7.0). Mix 200 ml of aqueous
         hydrochloric acid (1 mol/l) and 214 ml of aqueous
         tris(hydroxymethyl)aminomethane free base (121 g/l).

    2.   Sodium carbonate buffer (0.1 mol/l, pH 9.0). Mix 10 ml of aqueous
         sodium carbonate (10.6 g/l) and 890 ml of aqueous sodium
         bicarbonate solution (8.4 g/l).

    Standards

         Solutions containing theophylline concentrations of 5, 10, 20 and
    50 mg/l in blank plasma.

    Method

    1.   Add 0.5 ml of tris buffer to 2.0 ml of sample or standard, and
         add 10 ml of chloroform.

    2.   Vortex-mix for 2 minutes, and centrifuge for 5 minutes.

    3.   Remove the upper, aqueous layer and filter 8 ml of the chloroform
         extract through phase-separating filter-paper into a clean tube.

    4.   Add 2.5 ml of sodium carbonate buffer to the chloroform extract,
         vortex-mix for 2 minutes and centrifuge for 5 minutes.

    5.   Transfer 2.0 ml of the aqueous extract (upper layer) to a quartz
         spectrophotometer cell and measure the absorbance at 280 nm
         against a plasma blank (see section 4.5.2).

    Results

         Construct a calibration graph following analysis of the standard
    theophylline solutions and calculate the theophylline concentration in
    the sample. Specimens containing a theophylline concentration of more
    than 50 mg/l should be diluted with blank plasma and re-analysed.

         Use of the pH 9.0 extraction and measurement at 280 nm minimizes
    interference from barbiturates, but caffeine, theophylline metabolites
    and some other drugs may interfere in this assay.

    Sensitivity

         Theophylline, 5 mg/l.

    Clinical interpretation

         Acute overdosage with theophylline and other xanthines may cause
    palpitations, hypotension, diuresis, central nervous system
    stimulation, nausea, vomiting, marked hypokalaemia, metabolic acidosis
    and convulsions. Treatment is normally symptomatic and supportive,
    with particular emphasis on correcting the hypokalaemia. Repeat-dose
    oral activated charcoal can be used to enhance theophylline clearance
    (see section 2.2.3).

         Plasma theophylline concentrations attained during therapy are
    normally less than 20 mg/l. Toxic effects are more frequent at
    concentrations above 30 mg/l, while plasma concentrations of 50 mg/l
    or more may be encountered in fatal cases.

    6.106  Thiocyanates

         Potassium thiocyanate (KSCN) and sodium thiocyanate (NaSCN) were
    previously used in the treatment of hypertension, but nowadays these
    compounds are used principally as synthetic intermediates, and in the
    printing, dye and photographic industries. Thiocyanate is a metabolite
    of cyanide and thiocyanate toxicity is most commonly encountered as a
    result of chronic sodium nitroprusside administration. Thiocyanate is
    also found in the blood of cigarette smokers from metabolism of
    cyanide. Thiocyanate is excreted in urine; the plasma half-life is
    about 3 days if renal function is normal.

    Qualitative test

         Applicable to urine, stomach contents and scene residues.

    Reagent

         Aqueous ferric chloride solution (50 g/l).

    Method

         Add 0.1 ml of ferric chloride solution to 0.1 ml of sample and
    mix.

    Results

         A deep red colour indicates the presence of thiocyanate.

    Sensitivity

         Thiocyanate, 50 mg/l.

    Quantitative assay

         Applicable to plasma or serum.

    Reagents

    1.   Aqueous trichloroacetic acid solution (50 g/l).

    2.   Ferric nitrate reagent. Dissolve 80 g of ferric nitrate nona-
         hydrate in 250 ml of aqueous nitric acid (2 mol/l), dilute to
         500 ml with purified water and filter.

    Standards

         Prepare aqueous solutions containing thiocyanate ion
    concentrations of 5, 10, 20, 50 and 100 mg/l by dilution from an
    aqueous solution of potassium thiocyanate (1.67 g/l, equivalent to a
    thiocyanate ion concentration of 1.00 g/l).

    Method

    1.   Add 4.5 ml of trichloroacetic acid solution to 0.5 ml of sample
         or standard, vortex-mix for 30 seconds and centrifuge for 5
         minutes.

    2.   In a darkened room, add 2 ml of the supernatant to 4 ml of ferric
         nitrate reagent, vortex-mix for 5 seconds, and measure the
         absorbance at 460 nm against a reagent blank (see section 4.5.2).

    Results

         Construct a calibration graph following analysis of the standard
    thiocyanate solutions and calculate the thiocyanate concentration in
    the sample.

    Sensitivity

         Thiocyanate, 2 mg/l.

    Clinical interpretation

         Acute ingestion of thiocyanate salts may cause disorientation,
    weakness, hypotension, confusion, psychotic behaviour, muscular spasm
    and convulsions. Treatment is normally symptomatic and supportive.

         In nonsmokers, plasma thiocyanate concentrations range from 0.1
    to 0.4 mg/l, while in heavy smokers concentrations typically range
    from 5 to 20 mg/l. Plasma thiocyanate concentrations can reach
    100 mg/l during sodium nitroprusside therapy, but toxicity often
    occurs at concentrations above 120 mg/l. Plasma concentrations of the
    order of 200 mg/l have been reported in fatalities.

    6.107  Tin

         Metallic tin (Sn) and its inorganic salts are used in metallurgy
    and in tanning, polishing and metal coating ("tin" cans). Organotin
    compounds, usually ethyl, butyl or phenyl derivatives, such as
    tributyl tin, are used as pesticides, in antifouling paints on ships,
    and as stabilizers in plastics. Inorganic tin compounds are poorly
    absorbed after ingestion, but organotin derivatives are well absorbed
    from the gastrointestinal tract and can cause serious systemic
    toxicity.

         There are no simple qualitative tests for either inorganic tin or
    organotin compounds suitable for use in the diagnosis of acute
    poisoning.

    Clinical interpretation

         Ingestion of high doses of inorganic tin compounds may cause
    nausea, vomiting and diarrhoea. Acute exposure to organotin
    derivatives may cause headache, vomiting, abdominal pain, tinnitus,
    deafness, loss of memory, disorientation, coma and respiratory
    depression. Treatment is symptomatic and supportive.

    6.108  Tolbutamide

    1-Butyl-3- p-tolylsulfonylurea; C12H18N2O3S; relative molecular
    mass, 270

    CHEMICAL STRUCTURE 58

         Tolbutamide is a hypoglycaemic agent widely used to treat
    diabetes. About 85% of an oral dose is excreted in urine as the
    4-carboxy and 4-hydroxymethyl metabolites; only about 5% is excreted
    unchanged. The normal dose of this drug is up to 3 g/day; a fatality
    has followed the ingestion of 50 g of tolbutamide.

         There is no simple qualitative test for tolbutamide in biological
    specimens. However, the colorimetric procedure given below can be used
    to assess the severity of poisoning if overdosage is suspected. This
    method can also be used to measure other sulfonylurea hypoglycaemic
    agents, such as chlorpropamide and acetohexamide, in plasma using the
    appropriate standards.

    Quantitative assay

         Applicable to plasma or serum.

    Reagents

    1.   Aqueous hydrochloric acid (0.02 mol/l).

    2.   Fluorodinitrobenzene reagent. 1-Fluoro-2,4-dinitrobenzene (1 g/l)
         in  iso-amyl acetate, freshly prepared.

    Standards

         Solutions containing tolbutamide concentrations of 20, 50, 100
    and 200 mg/l in blank plasma.

    Method

    1.   Add 2.5 ml of dilute hydrochloric acid to 0.5 ml of sample or
         standard and add 10 ml of  iso-amyl acetate.

    2.   Vortex-mix for 2 minutes and centrifuge for 5 minutes.

    3.   Transfer 6 ml of the upper, organic layer to a clean tube, add 1
         ml of fluorodinitrobenzene reagent and vortex-mix for 30 seconds.

    4.   Place a loosely fitting glass cap on the top of the tube and heat
         on a boiling water-bath for 10 minutes.

    5.   Cool, allow to stand at room temperature for 30 minutes, and
         measure the absorbance at 346 nm against a plasma blank (see
         section 4.5.2).

    Results

         Construct a calibration graph following analysis of the standard
    tolbutamide solutions and calculate the tolbutamide concentration in
    the sample. Samples containing tolbutamide at concentrations above
    200 mg/l should be diluted with blank plasma and re-analysed.

         The chromogenic reagent used in this test reacts with most
    primary and secondary amines and some other functional groups. The
    results must therefore be interpreted with caution if other drugs may
    be present.

    Sensitivity

         Tolbutamide, 10 mg/l.

    Clinical interpretation

         Overdosage with sulfonylurea hypoglycaemics such as tolbutamide
    may cause nausea, vomiting, abdominal pain, hypotension, drowsiness,
    prolonged hypoglycaemia, hyperkalaemia, metabolic acidosis, coma,
    convulsions, pulmonary oedema and circulatory failure. Treatment
    includes correction of hypoglycaemia by giving glucose.

         Plasma tolbutamide concentrations attained during thera py are
    normally 40-100 mg/l and toxicity may be expected at plasma
    concentrations above 200 mg/l. Measurement of blood glucose is
    important in establishing the diagnosis of poisoning with tolbutamide
    and other hypoglycaemic drugs and in monitoring treatment (see section
    3.1.1).

    6.109  Toluene

    Methylbenzene; C6H5.CH3; relative molecular mass, 92

         Toluene is used as a solvent in adhesives, paints and paint-
    strippers (which often also contain dichloromethane or methanol) and
    is widely used in industry. Acute toluene poisoning is normally from
    massive accidental exposure or deliberate inhalation (glue sniffing,
    solvent abuse). Some 80% of a dose of toluene is metabolized to
    benzoic acid, which is conjugated with glycine to give hippuric acid.
    Measurement of urinary hippurate excretion can be used as an index of
    chronic toluene exposure, but sodium benzoate or benzoic acid used as
    food preservatives are also metabolized to hippurate and results
    should therefore be interpreted with caution.

    Quantitative assay

         Applicable to urine.

    Reagents

    1.   Aqueous hydrochloric acid (0.05 mol/l).

    2.   Dimethylaminobenzaldehyde reagent,  p-Dimethylaminobenzaldehyde
         (40 g/l) in acetic anhydride containing a few crystals (about
         0.5 g) of anhydrous sodium acetate.

    3.   Sodium chloride (solid).

    4.   Precipitated silica.

    Standards

         Blank urine, plus urine to which hippuric acid has been added to
    give concentrations of 0.2, 0.5, 1.0 and 2.0 g/l. All samples must be
    prepared from one urine specimen. These solutions are stable for 1
    month if stored at 4C in the dark.

    Method

    1.   Adjust the pH of 1.0 ml of sample or standard to 2 with dilute
         hydrochloric acid, and add sodium chloride until the solution is
         saturated.

    2.   Add 2 ml of diethyl ether:methanol (9:1), vortex-mix for 1 minute
         and centrifuge for 5 minutes.

    3.   Aspirate the upper, ether layer into a clean tube and re-extract
         the aqueous phase with a further 2 ml of diethyl ether:methanol
         (9:1).

    4.   Combine the ethereal extracts and add 1 ml to about 0.5 g of
         precipitated silica in a clean tube.

    5.   Remove the solvent under a stream of compressed air or nitrogen,
         add 3 ml of dimethylaminobenzaldehyde reagent, and heat on a
         heating block at 135C for 5 minutes.

    6.   Cool, add 4 ml of methanol, vortex-mix for 1 minute and
         centrifuge for 5 minutes.

    7.   Aspirate the methanolic extract into a clean tube and re-extract
         the silica with a further 4 ml of methanol.

    8.   Combine the methanolic extracts and measure the absorbance at
         460 nm against the blank urine extract (see section 4.5.2).

    Results

         Prepare a calibration graph following analysis of the standard
    hippuric acid solutions and calculate the hippuric acid concentration
    in the sample. It is important to use a portion of the same urine used
    to prepare the standards as the blank, since hippurate excretion
    varies with dietary benzoate intake, as indicated above.

    Sensitivity

         Hippurate, 0.1 g/l.

    Clinical interpretation

         Features of acute toluene poisoning include ataxia, nausea,
    vomiting, respiratory depression, coma and cardiac arrhythmia.
    Hepatorenal damage is uncommon. Treatment is symptomatic and
    supportive.

         Urinary hippuric acid concentrations are normally 0.1-0.2 g/l;
    concentrations greater than 1 g/l indicate prior exposure to toluene
    if other possible sources of benzoate can be excluded. In acute
    poisoning with toluene, death may supervene before hippurate excretion
    is raised.

    6.110  1,1,1-Trichloroethane

    Methylchloroform; CCl3.CH3; relative molecular mass, 133

         1,1,1-Trichloroethane is widely used as a solvent for dry-
    cleaning and vapour degreasing, and in typewriter correction fluids.
    Acute poisoning with 1,1,1-trichloroethane is normally from massive
    accidental exposure or deliberate inhalation (solvent abuse). Some 2%
    of an absorbed dose of 1,1,1-trichloroethane is metabolized to
    2,2,2-trichloroethanol and then to trichloroacetic acid. This can be
    detected in urine using the Fujiwara test.

    Qualitative test

         Applicable to urine. Fujiwara test - see carbon tetrachloride
    monograph (section 6.23).

    Results

         An intense red/purple colour in the upper, pyridine layer
    indicates the presence of trichloro compounds. The blank analysis
    excludes contamination with chloroform from the laboratory atmosphere.

         This test is sensitive and will detect ingestion or exposure to
    low doses of compounds that are extensively metabolized to
    trichloroacetic acid, such as chloral hydrate, dichloralphenazone
    and trichloroethylene, 12-24 hours later. However, with
    1,1,1-trichloroethane the test is correspondingly less sensitive,
    since such a small proportion of a dose is metabolized.

    Sensitivity

         Trichloroacetate, 1 mg/l.

    Clinical interpretation

         Signs of poisoning with 1,1,1-trichloroethane include ataxia,
    nausea, vomiting, coma, respiratory depression and cardiac
    arrhythmias. Hepatorenal damage is uncommon. Treatment is symptomatic
    and supportive.

    6.111  Trichloroethylene

    Trichloroethene; CHCl:CCl2; relative molecular mass, 131

         Trichloroethylene is a well known solvent and has also been used
    as a general anaesthetic. Acute poisoning with trichloroethylene is
    normally from massive accidental exposure or deliberate inhalation
    (solvent abuse). As with some hypnotic drugs, including chloral
    hydrate and dichloralphenazone, trichloroethylene is extensively
    metabolized (about 80% of an absorbed dose) to trichloroacetic acid.
    This can be detected in urine using the Fujiwara test.

    Qualitative test

         Applicable to urine. Fujiwara test - see carbon tetrachloride
    monograph (section 6.23).

    Results

         An intense red/purple colour in the upper, pyridine layer
    indicates the presence of trichloro compounds. The blank analysis
    excludes contamination with chloroform from the laboratory atmosphere.

         This test is very sensitive and will detect exposure to
    trichloroethylene 12-24 hours later. However, other compounds also
    give rise to trichloroacetic acid  in vivo, including the chlorinated
    solvents 1,1,1-trichloroethane and tetrachloroethylene, and caution
    must be exercised in reporting results.

    Sensitivity

         Trichloroacetate, 1 mg/l.

    Clinical interpretation

         Clinical features of acute poisoning with trichloroethylene
    include ataxia, nausea, vomiting, coma, respiratory depression and
    cardiac arrhythmias. Hepatorenal damage may also occur. Treatment is
    symptomatic and supportive.

    6.112  Verapamil

    5-( N-(3,4-Dimethoxyphenethyl)- N-methylamino)-2-(3,4-
    dimethoxyphenyl)-2-isopropylvaleronitrile; C27H38N2O4 relative
    molecular mass, 455

    CHEMICAL STRUCTURE 59

         Verapamil is used to treat hypertension. Metabolic pathways
    include  N-dealkylation ( N-demethylation gives norverapamil which
    is pharmacologically active) with  O-demethylation and conjugation of
    the resulting compounds. About 70% of a dose is excreted in urine, 10%
    as norverapamil, and less than 5% as the parent compound.

         There is no simple qualitative test for verapamil, but this
    compound and its metabolites can be detected and identified by thin-
    layer chromatography of a basic solvent extract of urine (see section
    5.2.3).

    Clinical interpretation

         Acute ingestion of verapamil may cause bradycardia, hypotension,
    cardiac arrhythmia, metabolic acidosis, hyperglycaemia, coma and
    gastrointestinal haemorrhage. Treatment is supportive, and may include
    the administration of calcium salts and inotropic agents in severe
    cases.

    6.113  Zinc

         Zinc (Zn) is used in some alloys (brass), in metal plating
    (galvanizing) and in many other applications. Finely divided zinc
    chloride (ZnCl2) is produced by chemical smoke generators and this
    compound is also used in soldering flux, dry battery cells and dental
    cement. Zinc oxide (ZnO) is used in making pharmaceuticals (zinc oxide
    plaster), rubber and white pigments. The acute oral toxicity of
    compounds like zinc chloride is limited since they are powerful
    emetics. Fatalities have been reported after the intravenous
    administration of 7.4 g of zinc and the inhalation of zinc chloride
    fumes.

         There is no simple qualitative or quantitative test for zinc in
    biological specimens.

    Clinical interpretation

         Acute poisoning with zinc compounds may cause fever, nausea,
    vomiting, diarrhoea, lethargy, muscle aches, weakness, cyanosis,
    pulmonary oedema, acute pancreatitis and acute renal failure.
    Treatment is symptomatic and supportive.

    Bibliography

     Section 1.  General considerations

    Baselt RC, Cravey RH.  Disposition of toxic drugs and chemicals 
          in man,  3rd ed. Chicago, Year Book Medical, 1990.

     Basic tests for pharmaceutical dosage forms. Geneva, World Health
         Organization, 1991.

     Basic tests for pharmaceutical substances. Geneva, World Health
         Organization, 1986.

    Duffus JH. Glossary for chemists of terms used in toxicology.
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    International Union of Pure and Applied Chemistry/International
         Programme on Chemical Safety.  Chemical safety matters. 
         Cambridge, Cambridge University Press, 1992.

    Moffat AC, ed.  Clarke's isolation and identification of drugs, 
         2nd ed. London, Pharmaceutical Press, 1986.

    Section 2.  Clinical aspects of analytical toxicology

    Dreisbach RH, Robertson WO.  Handbook of poisoning: prevention, 
          diagnosis and treatment, 12th ed. Norwalk, CT, Appleton Lange,
         1987.

    Ellenhorn MJ, Barceloux DG.  Medical toxicology: diagnosis and 
          treatment of human poisoning. Amsterdam, Elsevier, 1988.

    Goldfrank LR et al., ed.  Goldfrank's toxicologic emergencies, 4th ed.
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    Haddad LM, Winchester JF, ed.  Clinical management of poisoning and 
          drug overdose, 2nd ed. Philadelphia, Saunders, 1990.

    Meredith TJ et al., ed.  Naloxone, flumazenil and dantrolene as 
          antidotes. Cambridge, Cambridge University Press, 1993
         (IPCS/CEC Evaluation of Antidotes Series, Vol. 1).

    Meredith TJ et al., ed.  Antidotes for poisoning by cyanide.  
         Cambridge, Cambridge University Press, 1993 (IPCS/CEC Evaluation
         of Antidotes Series, Vol. 2).

    Meredith TJ et al., ed.  Antidotes for poisoning by paracetamol. 
         Cambridge, Cambridge University Press, 1994 (IPCS/CEC Evaluation
         of Antidotes Series, Vol. 3).

    Proudfoot AT.  Acute poisoning diagnosis and management, 2nd ed.
         Oxford, Butterworth Heinemann, 1993.

    Section 3.  General laboratory findings in clinical toxicology

    Walmsley RN, White GH.  A guide to diagnostic clinical chemistry.
         London, Blackwell, 1983.

    Whitehead TP et al.  Clinical chemistry and haematology: adult 
          reference values. London, BUPA, 1994.

    Section 4.  Practical aspects of analytical toxicology

    Anderson R.  Sample pretreatment and separation. Chichester, Wiley,
         1987 (Analytical chemistry by open learning (ACOL) series).

    Denney RC, Sinclair R.  Visible and ultraviolet spectroscopy.
         Chichester, Wiley, 1987 (Analytical chemistry by open learning
         (ACOL) series).

    Feldstein L, Klendhoj NC. The determination of volatile substances by
         microdiffusion analysis.  Journal of forensic sciences, 1957,
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    Hamilton RJ, Hamilton S.  Thin layer chromatography. Chichester, Wiley,
         1987 (Analytical chemistry by open learning (ACOL) series).

    Hawcroft D, Hector T.  Clinical specimens. Chichester, Wiley, 1987
         (Analytical chemistry by open learning (ACOL) series).

    Sewell P, Clarke B.  Chromatographic separations. Chichester, Wiley,
         1987 (Analytical chemistry by open learning (ACOL) series).

    Woodget BW, Cooper D.  Samples and standards. Chichester, Wiley, 1987
         (Analytical chemistry by open learning (ACOL) series).

    Section 5.  Qualitative tests for poisons

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         VCH, 1992.

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         the identification of drugs and poisons.  Analyst (London), 
         1982, 107:1106-1168.

    Section 6.  Monographs - analytical and toxicological data

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    Lenga RE.  The Sigma Aldrich library of chemical safety data, 2nd ed.
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          1,4-benzodiazepines in urine, blood and stomach contents.]
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         Crop Protection Council, 1991.

    Glossary

    This glossary is included to aid communication between the
    toxicologist and the clinician. The definitions given refer to the use
    of terms in this book, and are not necessarily valid in other
    contexts.

     Abortifacient  A means of causing abortion.

     Abuse  Excessive or improper use of drugs or other substances.

     Abuse, volatile substance  The intentional inhalation of volatile
         substances, such as organic solvents or aerosol propellants, with
         the aim of achieving intoxication.

     Acetylcholine  The main neurotransmitter of the vertebrate and
         invertebrate peripheral nervous systems ( see also: 
         Anticholinergic, Cholinergic).

     Acetylcholinesterase  Acetylcholine acetylhydrolase, EC 3.1.1.7.
         Enzyme that hydrolyses the neurotransmitter acetylcholine within
         the central nervous system ( see also: Cholinesterase).

     Acidosis  Pathological condition resulting from accumulation of acid
         in, or loss of base from, the blood or body tissues.

     Acidosis, lactic  Metabolic acidosis due to the production of
         excessive amounts of lactic acid.

     Acidosis, metabolic  Acidosis of metabolic origin.

     Acidosis, respiratory  Acidosis of respiratory origin.

     Acne Inflammation in or around the sebaceous glands, generally of the
         face, chest and back.

     Acute  Sudden or short-term (cf. Chronic).

     Acute-on-chronic  Describes a sudden episode of increased severity
         against a background of prolonged disease or exposure.

     -Adrenoceptor blocking agent  See -Blocker.

     Agonist  Drug that has affinity for, and stimulates physiological
         activity at, cell receptors (cf. Antagonist).

     -Agonist  Agent exerting an agonist effect at a -adrenoceptor.

     Agranulocytosis  A blood disorder in which there is an absence of
         granulocytes.

     Akathisia  An inability to sit still.

     Albuminuria  The presence of albumin in the urine.

     Alkaline diuresis  Technique for rendering the urine alkaline, for
         example, by intravenous administration of sodium bicarbonate, to
         enhance excretion of certain acidic poisons such as salicylate.

     Alkalinization  To add alkali or to make alkaline.

     Alkaloid  A nitrogenous organic compound of plant origin.

     Alkalosis  Pathological condition resulting from accumulation of base
         in, or loss of acid from, the blood or body tissues.

     Anaemia  Deficiency of erythrocytes or of haemoglobin in the blood.

     Anaesthetic  A substance producing either local or general loss of
         sensation.

     Analgesic  A substance that relieves pain, without producing
         anaesthesia or loss of consciousness.

     Anaphylaxis  Reaction to foreign material as a result of increased
         susceptibility following previous exposure.

     Angioedema  Marked swelling of body tissues.

     Anion gap  In blood plasma, the difference between the concentration
         of sodium and the sum of the concentrations of chloride and
         bicarbonate.

     Anorexia  Lack or loss of appetite for food (cf. Bulimia).

     Anoxia  Absence or lack of oxygen.

     Antagonist  An agent that reverses or reduces the pharmacological
         action of a second agent.

     Anthelminthic  An agent that kills intestinal worms.

     Antiarrhythmic  An agent used to treat a cardiac arrhythmia.

     Antibiotic  An agent produced or derived from a microorganism used to
         control or kill other microorganisms.

     Antibody  A protein produced in the body in response to exposure to an
         antigen; it recognizes and specifically binds the antigen.

     Anticholinergic  An antagonist to the neurotransmitter acetylcholine.

     Anticoagulant  A drug that prevents clotting of blood.

     Anticonvulsant  A drug used to control epilepsy.

     Antidepressant  A drug used to treat depression.

     Antidiabetic  A drug used to treat diabetes mellitus.

     Antidote  An agent that neutralizes or opposes the action of a poison
         on an organism.

     Antigen  Any substance that stimulates the body to produce an
         antibody.

     Antihistamine  An antagonist to histamine.

     Anti-inflammatory  Reducing or preventing inflammation.

     Anti-knock agent  A substance, such as tetra-ethyl lead, used to
         prevent pre-ignition (knock) in internal combustion engines.

     Antipsychotic  A drug used to treat psychosis.

     Antipyretic  A drug that relieves or reduces fever.

     Antiseptic  An agent used to control or kill microorganisms.

     Anuria  Complete absence of urine production (cf. Oliguria, Polyuria).

     Apathy  Indifference.

     Apnoea  Cessation of breathing.

     Areflexia  Generalized absence of reflexes.

     Arrhythmia  Any variation from the normal rhythm of the heartbeat.

     Aspiration  (i) The act of withdrawing a fluid by suction. (ii) The
         breathing in of a foreign body, e.g. vomit.

     Asthma  Chronic respiratory disease characterized by wheezing and
         difficulty in breathing out.

     Ataxia  Failure of muscular coordination.

     Bilirubin  A pigment, derived from the breakdown of haemoglobin, that
         occurs in soluble form in blood and in bile.

     Biological specimens  Samples of tissues (including blood, hair),
         secretions (breast milk, saliva, sweat), excretion products
         (bile, urine) and other material such as stomach contents or
         vomit derived from a patient.

     Blank  Used in analytical chemistry to denote a specimen not
         containing the analyte of interest and from which a background
         reading can be obtained.

     -Blocker  Agent inhibiting the action of endogenous neurotransmitters
         (epinephrine, norepinephrine) at -adrenoceptors.

     Bradyarrhythmia  Arrhythmia associated with an excessively slow
         heartbeat (cf. Tachyarrhythmia).

     Bradycardia  Excessively slow heartbeat (cf. Tachycardia).

     Bronchoconstriction  Narrowing of the bronchial tubes.

     Bronchodilation  Expansion of the bronchial tubes.

     Bronchorrhoea  Abnormally copious mucous discharge from the walls of
         the bronchial tubes.

     Bronchospasm  Intermittent, violent contraction of the walls of the
         bronchial tubes.

     Bulimia  Morbid hunger (cf. Anorexia).

     Butyrophenones  A group of antipsychotic drugs.

     Carboxyhaemoglobin  Product formed when carbon monoxide binds to
         haemoglobin.

     Cardiogenic  Produced in, or originating from, the heart.

     Cardiotoxic  Having a harmful effect on the action of the heart.

     Catheterization  Introduction of a tube for adding or removing
         fluids to or from the body.

     Caustic  Having a corrosive action on skin and flesh.

     Cerebellar  Relating to the hind part of the brain concerned with
         voluntary movement and balance.

     Cerebral  Relating to the brain.

     Chelate  Compound in which a central metallic ion is attached to an
         organic molecule (chelating agent) at two or more positions
         ( see also: Sequestrant).

     Chelating agent  A compound capable of forming a chelate with a metal
         ion.

     Chelation therapy  Treatment with a chelating agent to enhance the
         elimination or reduce the toxicity of a poison.

     Cholinergic  Stimulated, activated or transmitted by acetylcholine.

     Cholinesterase  Enzyme (E.C. 3.1.1.8) that catalyses the breakdown of
         a choline ester to choline ( see also: Acetylcholinesterase).

     Chorea  Irregular, involuntary movements of the limbs or face.

     Chronic  Long-term (cf. Acute).

     Cirrhosis  Wasting disease of the liver accompanied by abnormal growth
         of connective tissue (scar tissue).

     Coagulopathy  Disorder of blood clotting.

     Colic  Severe, intermittent pain associated with the abdomen.

     Conjugate  Metabolite formed by covalent bonding with, for example,
         glucuronic acid, sulfate, acetate, or glycine.

     Conjunctiva  The outer surface of the eyeball and the inner surface of
         the eyelid.

     Conjunctivitis  Inflammation of the conjunctiva.

     Contaminant  An impurity.

     Corrosive  Able to eat away or dissolve by chemical action.

     Cosmetic  Concerned with improving appearance or hygiene.

     Cross-contamination  Accidental introduction of an impurity.

     Crystalluria  Presence of crystals in the urine.

     Cutaneous  Associated with the skin.

     Cyanosis  Blue appearance, especially of the skin and mucous
         membranes, due to deficient oxygenation.

     Deamination  Removal of an amine from a molecule.

     Delirium  State characterized by hallucinations, disorientation, and
         restlessness.

     Delirium tremens  Clinical features associated with alcohol withdrawal
         ( see also:  Withdrawal, drug).

     Denature  (i) To alter the physical nature of a substance or mixture.
         (ii) To render unfit for human consumption.

     Depigmentation  Loss of natural coloration.

     Depilatory agent  A substance applied topically to remove unwanted
         hair.

     Depression, respiratory  (i) Abnormally low rate and depth of
         breathing. (ii) Reduction in the amount of oxygen available to
         tissues ( see also: Hypoxia).

     Derivative  A substance formed from a primary compound by chemical
         reaction.

     Dermal  Relating to the skin.

     Dermatitis  Inflammation of the skin.

     Dermatitis herpetiformis  Disease characterized by the irregular
         occurrence of groups of intensely irritating skin lesions, the
         sites of which eventually become pigmented.

     Descaling agent  Substance used to remove deposits from kettles and
         other vessels.

     Detergent  A chemical cleaning agent.

     Diabetes mellitus  Disorder of glucose metabolism due to insulin
         deficiency.

     Dialysis  The separation of substances by diffusion through a
         semipermeable membrane.

     Dialysis, peritoneal  Procedure whereby blood is purified by dialysis
         against fluid infused into the peritoneal cavity and subsequently
         removed. The aim is to remove unwanted compounds of low relative
         molecular mass from the circulation.

     Diluent  A fluid used in dilution.

     Diplopia  Double vision.

     Discriminating power  The ability of a system to distinguish between a
         number of possibilities.

     Disinhibition  Removal of restraints on behaviour.

     Disorientation  Confused as to direction.

     Disseminated intravascular coagulation  Blood clotting throughout
         the systemic circulation, but associated with abnormal bleeding.

     Diuresis  Increased production of urine.

     Diuresis, forced  Abnormally enhanced urine production, for example
         following administration of intravenous fluids or diuretics.

     Diuretic  An agent that increases urine production.

     Drug  A substance that, when administered to an organism or a system
         derived from an organism, may modify one or more of its
         functions.

     Drug, controlled  A drug whose use is regulated by law.

     Dysphagia  Difficulty in swallowing.

     Dyspnoea  Difficult or laboured breathing.

     Dystonic reaction  A consequence of an alteration in the tone of a
         tissue.

     Elimination half-life. See Plasma half-life.

     Embalm  To preserve a body after death.

     Emesis  Vomiting.

     Emetic  Substance causing emesis.

     Encephalopathy  Degenerative brain disease.

     Enteral  Within the intestine; usually used to refer to oral
         administration of an agent.

     Enterohepatic recirculation  A cycle in which substances excreted in
         bile are reabsorbed from the intestine.

     Epigastric  Concerned with the part of the abdomen extending from the
         sternum to the umbilicus (epigastrum).

     Erythrocyte  Red blood cell.

     Euphoria  An exaggerated feeling of well-being.

     Euthanasia  The bringing about of a gentle and easy death in the case
         of incurable and painful disease.

     Fibrillation, ventricular  Serious loss of coordination in the
         contraction of the muscle fibres of the ventricles of the heart,
         leading to cessation of blood flow from the heart.

     Fibrosis  The development of abnormal connective tissue, usually as
         a response to injury.

     First-pass metabolism  The fraction of an oral dose metabolized in
         the liver or gut wall before reaching the systemic circulation.

     Fumigant  A vapour used to kill pests.

     Fungicide  A pesticide used to kill fungi or check the growth of
         spores.

     Gag reflex  Automatic response which normally prevents inhalation of
         vomit by closing the epiglottis, the cartilaginous flap over the
         trachea.

     Gastric  Relating to the stomach.

     Gastritis  Inflammation of the stomach.

     Gastroenteritis  Inflammation of the lining of the stomach and
         intestine.

     Gastrointestinal  Relating to the stomach and intestine.

     Genitourinary  Relating to the genitalia and the urinary system.

     Granulocyte  A type of white blood cell.

     Haematemesis  Vomiting of blood.

     Haematocrit  Erythrocyte volume fraction; the ratio by volume of the
         blood cells to plasma.

     Haematoma  Swelling composed of blood.

     Haematuria  Blood in the urine.

     Haemodialysis  Procedure whereby blood is dialysed against a large
         volume of isotonic fluid outside the body and then returned to
         the systemic circulation. Used to remove unwanted compounds of
         low relative molecular mass from the circulation.

     Haemoglobin  An iron-containing pigment found in erythrocytes, which
         binds oxygen for transport within the bloodstream.

     Haemolysis  Rupture of erythrocytes leading to the appearance of free
         haemoglobin in the plasma.

     Haemoperfusion  Procedure whereby blood is passed through a column of
         adsorbent material outside the body and then returned to the
         systemic circulation. Used to remove unwanted components of low
         relative molecular mass from the circulation.

     Haemorrhage  Bleeding.

     Haemostasis  Stoppage of bleeding.

     Halide  A compound consisting of halogen ions together with metallic
         or organic counter-ions.

     Hallucination  An imagined occurrence, either visual or auditory.

     Hallucinogen  A substance causing a hallucination.

     Halogen  A member of the series of elements consisting, for practical
         purposes, of fluorine, chlorine, bromine and iodine.

     Headspace  The space above a solid or liquid in a container.

     Hepatic  Relating to the liver.

     Hepatitis  Inflammation of the liver.

     Hepatorenal  Relating to the liver and kidneys.

     Hepatotoxic  Harmful to the liver.

     Herbicide  A pesticide used to control or kill plants or plant seeds.

     Histamine  An amine present in many tissues, release of which can
         cause dilatation of the capillary blood vessels, flushing and
         other effects.

     Hydrolysis  Decomposition caused by or involving water.

     Hydrophilic  Readily soluble in water.

     Hydrophobic  Not readily soluble in water.

     Hyperactive  Abnormally active.

     Hyperbilirubinaemia  An excess of bilirubin in the blood.

     Hypercalcaemia  Abnormally high blood calcium concentration.

     Hyperglycaemia  Abnormally high blood sugar (glucose) concentration.

     Hyperkalaemia  Abnormally high blood potassium concentration.

     Hypernatraemia  Abnormally high blood sodium concentration.

     Hyperpnoea  Abnormally rapid and deep breathing ( see also: 
         Hyperventilation, Tachypnoea).

     Hyperpyrexia  Abnormally high body temperature.

     Hyperreflexia  Abnormally exaggerated reflexes.

     Hypersalivation  Excessive production of saliva.

     Hypertension  Abnormally high blood pressure.

     Hyperthermia  Dangerously high body temperature.

     Hyperventilation  Increased rate and depth of respiration
         ( see also Hyperpnoea).

     Hypnotic  Capable of inducing sleep.

     Hypocalcaemia  Abnormally low blood calcium concentration.

     Hypoglycaemia  Abnormally low blood sugar (glucose) concentration.

     Hypokalaemia  Abnormally low blood potassium concentration.

     Hypophosphataemia  Abnormally low blood phosphate concentration.

     Hypostatic  Caused by the combined effects of gravity and poor
         circulation of the blood.

     Hypotension  Abnormally low blood pressure.

     Hypothermia  Abnormally low body temperature.

     Hypotonia  Abnormally low muscle tone.

     Hypoxia  Reduction of oxygen in an animal body below physiological
         requirements ( see also: Anoxia; Depression, respiratory).

     Iatrogenic  Induced in a patient by the comments or treatment of a
         physician. Used especially in connection with inappropriate drug
         treatment.

     Incontinence  Lack of voluntary control over the discharge of urine or
         faeces.

     Inebriation  Excitement or elation induced by alcohol or other drugs.

     Inflammation  Soreness and pain in joints or other parts of the body.

     Ingestion  Taking of substances into the body by mouth.

     Inotrope  An agent that increases or decreases the contractility of
         the heart muscle.

     Insecticide  A pesticide used to control or kill insects.

     Inspiration  The act of breathing in.

     Intoxication  (i) Poisoning. (ii) Inebriation.

     Ischaemia  Deficiency of blood supply to a part of the body.

     Isobestic point  Wavelength at which the specific absorbances of two
         interconvertible materials are the same, regardless of the
         equilibrium position of the reaction between them.

     Isotonic  Having the same osmolality.

     Jaundice  Disease characterized by the deposition of yellow bile
         pigments in, for example, the eyes and skin.

     Ketoacidosis  Metabolic acidosis due to the production of excessive
         amounts of ketones such as acetone.

     Ketonuria  The presence of excessive amounts of ketones, such as
         acetone, in urine, often indicating a disorder of glucose
         metabolism, such as diabetes mellitus, but sometimes due simply
         to starvation.

     Lacrimation  The secretion of tears.

     Leishmaniasis  Disease caused by infection with a protozoon
         transmitted to humans by sandflies.

     Leukocyte  White blood cell.

     Leukocyte count  Concentration of white blood cells in a sample of
         blood.

     Limit of detection  The smallest amount of a substance that can be
         revealed by a test carried out in a prescribed manner.

     Lipaemia  The presence of abnormal amounts of fats in the blood.

     Lipophilic  Readily soluble in fats and organic solvents.

     Lipophobic  Not readily soluble in fats and organic solvents.

     Maintenance therapy  Planned, long-term drug therapy; for example,
         treatment of opiate dependence with methadone.

     Malaise  Feeling of discomfort or sickness.

     Mania  Mental illness characterized by elation, excessively rapid
         speech and violent, destructive actions.

     MAOI  See Monoamine oxidase inhibitor.

     Metabolism  Chemical reactions occurring in organisms or in systems
         derived from organisms, whereby the function of nutrition is
         effected.

     Metabolite  A substance produced by metabolism.

     Methaemoglobin  Oxidized haemoglobin.

     Methaemoglobinaemia  The presence of abnormal amounts of oxidized
         haemoglobin in the blood.

     Miosis  Contraction of the pupil of the eye (cf. Mydriasis).

     Mixer, rotary  A device for mixing solutions or suspensions by means
         of a gentle rotating motion. Used for solvent extraction or other
         procedures requiring mixing of relatively large quantities of
         material (cf. Vortex-mixer).

     Monoamine oxidase inhibitor  An antidepressant that acts by
         inhibiting the metabolism of amines acting as neurotransmitters
         in the brain.

     Mydriasis  Extreme dilatation of the pupil of the eye.

     Myocardial  Relating to the myocardium, the muscle of the heart.

     Myoclonus  A sudden shock-like muscular contraction which may
         involve one or more muscles or a few fibres of a muscle.

     Myoglobin  A protein related to haemoglobin, found in muscle.

     Myoglobinuria  The presence of myoglobin in the urine.

     Narcotic  An agent that produces insensibility or stupor (narcosis).

     Nausea  A feeling of need to vomit.

     Necrosis  Cell death due to anoxia or local toxic or microbiological
         action. Used particularly to describe cell death at a focal point
         in a multicellular organism.

     Neonatal  Newly born.

     Nephrotoxic  Harmful to the kidney.

     Neuroleptic  A drug that produces analgesia, sedation and
         tranquillization; used in the treatment of psychosis.

     Neuropathy, peripheral  Disease characterized by disintegration or
         destruction of the specialized tissues of the peripheral nervous
         system.

     Neuropsychiatric  Relating to the nervous system and mental
         processes.

     Neurotoxic  Harmful to nerve tissue.

     Neurotransmitter  Compound, e.g. acetylcholine, responsible for
         transmission of nerve impulses at synapses.

     Nystagmus  Constant, involuntary, jerky eye movement.

     Ocular  Relating to the eye.

     Oedema  Pathological accumulation of fluid in tissue spaces.

     Oliguria  Production of a diminished amount of urine (cf. Anuria,
         Polyuria).

     Ophthalmic  Relating to the eye.

     Opiate  A pharmacologically active agent, such as morphine, derived
         from opium.

     Opioid  An agent that has agonist activity at specific receptors in
         the brain.

     Opium  The dried juice of the poppy  Papaver somniferum.

     Opisthotonos  Extreme arching backwards of the spine and neck as a
         result of muscle spasm.

     Osmolality  The osmotic strength of a solution.

     Osmotic  Relating to osmosis.

     Osteomalacia  Softening of bones as a result of loss of calcium
         salts.

     Palpitations  Abnormal awareness of the heartbeat.

     Pancreatitis  Inflammation of the pancreas.

     Papilloedema  Oedema of the optic nerve-head.

     Paraesthesia  Numbness and tingling.

     Paralysis  Loss of power in any part of the body.

     Paralytic ileus  Distension of the intestine due to paralysis of the
         muscle of the intestinal wall.

     Parenteral  By some means other than through the intestinal canal.
         Usually used to refer to intramuscular, intraperitoneal or
         intravenous administration of a substance.

     Parkinsonism  A syndrome characterized by muscle rigidity, hand
         tremor, mask-like facial expression and other features.

     Parotid gland  Salivary gland near to the ear.

     Perinatal  In humans, relating to the period between the
         twenty-second week of pregnancy and the first week after birth.

     Pesticide  Substance used to kill or control any pest, including
         animals, plants, fungi, or other organisms in agricultural,
         industrial and domestic situations.

     Petechiae  Small red or purple spots caused by accumulation of blood
         beneath the skin.

     Pharmacokinetics  The action of drugs in the body over a period of
         time, including the processes of absorption, distribution,
         metabolism and elimination.

     Phenylketonuria  Inherited disorder of phenylalanine metabolism
         characterized by the appearance of phenylpyruvic acid in the
         urine.

     Phosphorylation, oxidative  Electron transport process whereby
         energy released by oxidation of products from the tricarboxylic
         acid cycle in mitochondria is stored initially as adenosine
         triphosphate.

     Pin-point pupils  Extreme contraction of the pupils of the eyes
         ( see also Miosis).

     Pipette, automatic  Device used to dispense repeatedly known volumes
         of a fluid.

     Pipette, positive-displacement  Device with a washable tip, used to
         take up and dispense known volumes of a fluid, and in which the
         plunger is in contact with the fluid. Used to dispense viscous
         solutions such as whole blood.

     Pipette, semi-automatic  Device, often with disposable tips, used to
         take up and dispense known volumes of aqueous fluids such as
         plasma or serum. Reliable only for fluids with a viscosity
         similar to that of water.

     Plasma  The fluid portion of circulating blood (cf. Serum).

     Plasma half-life  The time taken for the plasma concentration of a
         substance to decrease by half.

     Platelet count  The concentration of platelets in a sample of blood.

     Pneumonitis  Inflammation of the lung.

     Poison  A chemical that may harm or kill an organism.

     Polyuria  Production of an excessive amount of urine (cf. Anuria,
         Oliguria).

     Prophylaxis  Treatment intended to prevent the occurrence of
    disease.

     Protective agent  Substance that can prevent the manifestations of
         toxicity of an agent on an organism (cf. Antidote).

     Protein binding  Non-covalent adherence of drugs and other agents to
         protein. In plasma, acidic compounds normally bind to albumin and
         bases may also bind to alpha1-acid glycoprotein.

     Proteinaemia  The presence of excessive amounts of protein in blood.

     Prothrombin time  A measure of the time taken for blood to clot
          in vitro. Often reported as a ratio to a control (normal)
         value.

     Psychoactive  Affecting the brain and influencing behaviour;
         psychotropic.

     Psychosis  A serious mental disorder characterized by confusion,
         delusions, and hallucinations.

     Psychotropic  Affecting the brain and influencing behaviour;
         psychoactive.

     Pulmonary  Relating to the lungs.

     Putrefaction  Process of decomposition occurring in dead tissue.

     Pyrexia  Raised body temperature; fever.

     Reconstitute  Redissolve a solute after removal of a solvent.

     Relative density  The ratio of the density of a material to the
         density of a reference material, usually water.

     Renal  Relating to the kidneys.

     Repellent  A substance used to drive away pests, such as insects.

     Rhabdomyolysis  Muscle breakdown leading to the appearance of
         myoglobin in blood and urine.

     Rodenticide  Pesticide used to control or kill rats or other rodents.

     Rubefacient  Causing reddening of the skin.

     Salicylism  Chronic poisoning caused by excessive use of salicylates.
         Characterized by respiratory alkalosis followed by metabolic
         acidosis.

     Scene residue  Material found at the scene of a poisoning incident.

     Schistosomiasis  Infection with trematode parasitic flukes of the
         genus  Schistosoma.

     Schizophrenia  A form of psychosis in which there are fundamental
         distortions of thinking and perception. Delusions and
         hallucinations are common clinical features.

     Screening  (i) In clinical toxicology, a search for unknown poisons
         by chemical analysis of biological or other specimens (drug
         screen, poisons screen). (ii) In experimental toxicology, a
         search for possible toxicity of a substance in normal use (safety
         screen).

     Sedative  An agent that quiets nervous excitement; used to treat
         agitation.

     Sensitivity  An indication of the minimum quantity of a substance
         that can be detected and identified by a test.

     Sequelae  Consequences of disease or injury.

     Sequestrant  A substance that removes an ion or renders it
         ineffective ( see also: Chelate).

     Serum  (i) The clear, usually watery, fluid that moistens the
         surface of internal membranes. (ii) The watery portion of blood
         which remains after blood clots (cf. Plasma).

     Shock  The general metabolic and other consequences of severe
         injury, characterized by low body temperature, low blood
         pressure, rapid pulse, pale, cold, moist skin and, often,
         vomiting, restlessness and anxiety.

     Sign  Objective evidence of disease or of an effect induced by a
         poison, perceptible to an examining physician (cf. Symptom).

     Spike  In analytical chemistry, to add a known amount of a pure
         compound to a blank specimen to act as a positive control.

     Spray reagent  See Visualization reagent.

     Stasis  Stoppage.

     Stellate ganglion block  Procedure in which local anaesthesia is
         induced in the branch of the inferior cervical ganglion concerned
         with vision.

     Stimulant  An agent that increases activity, for example in the
         central nervous system.

     Stomatitis  Inflammation of the mucous membrane of the mouth.

     Stupor  Lethargy; torpor; unconsciousness.

     Subclinical  Describes changes resulting from disease or intoxication
         that do not produce clinically recognizable symptoms.

     Sublingual  Beneath the tongue.

     Submaxillary gland  Salivary gland situated beneath the lower jaw.

     Supernatant  An upper layer of liquid.

     Sympathomimetic  A drug that mimics the action of endogenous
         neurotransmitters in the sympathetic nervous system.

     Symptom  Subjective evidence of disease or intoxication as perceived
         by the patient (cf. Sign).

     Synapse  Area of contact between two nerve cells.

     Syncope  Loss of consciousness caused by a sudden fall of blood
         pressure in the brain.

     Synergist  A substance that increases the effect of another.

     Systemic  Affecting the body as a whole.

     Tachyarrhythmia  An arrhythmia associated with an excessively rapid
         heartbeat.

     Tachycardia  Excessively rapid heartbeat.

     Tachypnoea  Unduly rapid breathing (cf. Hyperpnoea).

     Tetany  Heightened excitability of the motor nerves with painful
         muscle cramps.

     Thrombocytopenia  Abnormally low number of platelets in the blood.

     Tinnitus  A continual noise in the ears, such as ringing, buzzing,
         roaring or clicking.

     Tolerance  (i) The ability of an organism to experience exposure to
         potentially harmful amounts of a poison without showing evidence
         of toxicity. (ii) An adaptive state whereby the pharmacological
         effects of the same dose of a substance become diminished as a
         result of repeated exposure.

     Toxin  A poison of natural origin.

     Toxic  Able to cause injury to living organisms as a result of
         chemical interaction within the organism.

     Toxicity  Any harmful effect of a chemical on an organism.

     Toxicology  The study of the actual or potential danger to organisms
         presented by the harmful effects of chemicals.

     Tranquillizer  A drug used to treat anxiety.

     Tremor  Shaking or quivering, especially in the hands.

     Tricarboxylic acid cycle  Citric acid cycle, Krebs cycle: intermediary
         metabolic sequence whereby energy from fats and sugars is made
         available for oxidative phosphorylation, among other functions.

     Tricyclic antidepressant  Drug, such as amitriptyline, characterized
         by the presence of three conjugated aromatic rings and used to
         treat depression.

     Tuberculosis  A disease caused by  Mycobacterium tuberculosis, 
         characterized by the development of nodules (tubercules) in
         affected tissues, for example the lung.

     Ulcer, peptic  An ulcer of the stomach or duodenum.

     Ulceration  Formation of open sores.

     Uraemia  The clinical state arising from kidney failure; literally,
         excess urea in the blood.

     Urinary retention  Abnormal retention of urine in the bladder.

     Urticaria  An acute or chronic dermatitis characterized by the
         presence of white, red or pink spots on the skin accompanied by
         itching, stinging or burning sensations.

     Vasodilatation  Dilatation (expansion) of a blood vessel leading to
         increased flow of blood through the vessel.

     Vehicle  A substance with which a drug or other substance is mixed
         for administration or application.

     Vertigo  A sensation of dizziness, leading to loss of balance.

     Viscosity  (of a fluid) Resistance to flow.

     Visualization reagent  A substance or solution used to reveal the
         presence of other substances on thin-layer chromatograms, for
         example.

     Volume of distribution  Theoretical relationship, useful as an
         indicator of tissue distribution, between the amount of a drug or
         other compound in the body ( d mg) and the concentration of the
         compound measured in whole blood or in plasma/serum ( c mg/l):

         Volume of distribution =  d/c litres.

         Water-soluble compounds such as phenazone (see section 6.39) have
         a volume of distribution comparable to the volume of water in the
         body, while lipophilic compounds such as digoxin (see section
         6.41) have a volume of distribution many times greater.

          Relative volume of distribution  is volume of distribution
         divided by body weight.

     Vortex-mixer  A device for mixing solutions or suspensions by means of
         a whirling motion which creates a cavity in the centre of the
         mixture. Used for solvent extraction and other procedures
         requiring efficient mixing of relatively small quantities of
         material (up to about 10 ml total volume) (cf. Mixer, rotary).

     Withdrawal, drug  The act or consequences of reduction or cessation of
         drug administration in a dependent subject. The clinical features
         observed (commonly sweating, tremor, nausea, vomiting) are often
         reversible if drug use is recommenced ( see also  Delirium
         tremens).

     Xenobiotic  Compound foreign to the metabolism of an organism.

    Annex 1

    List of reference compounds and reagents

         The reference compounds and reagents required for the tests are
    listed here. A number are controlled drugs and special arrangements
    are necessary to obtain and store these items (see section 1.2). Where
    appropriate, the commonly encountered salts of basic drugs and some
    other compounds have been indicated. Compounds marked with an asterisk
    (*) are used both as reagents and reference compounds.

    Reference compounds

     Pharmaceuticals and metabolites 

     N-Acetylprocainamide (hydrochloride)
    Acetylsalicylic acid
    Amitriptyline (hydrochloride)
    Amobarbital
    Amfetamine (sulfate)
    Atropine (sulfate)

    Barbital (sodium)
    Benzoylecgonine
    Bisacodyl
    Brucine (sulfate)

    Caffeine
    Carbamazepine
    Chloroquine (phosphate)
    Chlorpromazine (hydrochloride)
    Clomethiazole (ethandisulfonate)
    Clomipramine (hydrochloride)
    Cocaine
    Codeine (phosphate)
    Cyclizine (hydrochloride)

    Dantron
    Dapsone
    Desipramine (hydrochloride)
    Dextropropoxyphene (hydrochloride)
    Digoxin
    Digitoxin
    Dihydrocodeine (tartrate)
    Diphenhydramine (hydrochloride)
    Dosulepin (hydrochloride)
    Doxepin (hydrochloride)

    Emetine (hydrochloride)
    Ephedrine (hydrochloride)
    Ethchlorvynol

    Flurazepam (hydrochloride)

    Glutethimide
    Glyceryl trinitrate (aqueous solution)

    Haloperidol

    Imipramine (hydrochloride)*
    Isoniazid

    Lidocaine (hydrochloride)
    Lithium (carbonate)

    Meprobamate
    Methadone (hydrochloride)
    Methaqualone
    Metamfetamine (hydrochloride)
    Methyprylon
    Monoacetyldapsone
    Morphine (sulfate)
    Morphine-3-glucuronide

    Nitrazepam
    Nortriptyline (hydrochloride)

    Orphenadrine (citrate)

    Paracetamol
    Pentazocine (hydrochloride)
    Perphenazine
    Pethidine (hydrochloride)
    Phenazone
    Phenobarbital
    Phenolphthalein
    Phenytoin
    Pralidoxime (chloride)*
    Procainamide (hydrochloride)
    Propranolol (hydrochloride)

    Quinidine (sulfate)
    Quinine (sulfate)*

    Rhein

    Salicylic acid
    Strychnine (hydrochloride)

    Theophylline
    Thioridazine (hydrochloride)
    Trifluoperazine (hydrochloride)
    Trimipramine (maleate)
    Tolbutamide

    Verapamil (hydrochloride)

     Pesticides 

    Aldicarb
    Aldrin

    Bromophos
    Bromoxynil

    Carbaryl
    Chloralose
    Chlorpyrifos

    2, 4-D
    DNOC
    2, 4-DP
    Dicophane
    Dimethoate
    Dinoseb
    Dioxathion
    Diquat

    Heptachlor

    Ioxynil

    Lindane

    MCPA
    MCPP
    Methidathion
    Methiocarb

    Nicotine

    Paraquat
    Pentachlorophenol
    2, 4, 5-T
    2, 4, 5-TP

    Reagents and solvents

    Acetaldehyde
    Acetamide
    Acetic acid (glacial)
    Acetic anhydride
    Acetone
    Acetylthiocholine iodide
    Alcohol dehydrogenase (yeast, crystalline)
    Alizarin complexone
    2-Aminoethanol
    1-Aminonaphthalene
    Ammonium acetate
    Ammonium chloride
    Ammonium hydroxide (concentrated, relative density 0.88)
    Ammonium molybdate
    Ammonium sulfamate
    Ammonium sulfate
    Ammonium thiocyanate
    Ammonium vanadate (finely powdered)
     iso-Amyl acetate
    Arsenic trichloride

    Barbituric acid (malonylurea, 2,4,6-trihydroxypyrimidine)
    Basic magenta (fuchsine, CI 42510)
    2,2'-Bipyridyl (2,2'-dipyridyl)
    Boric acid*
    Bromine
    Butanone (methyl ethyl ketone)
     n-Butyl acetate

    Calcium chloride
    Calcium hydroxide
    Calcium hypochlorite (powdered)
    Carbon monoxide* (or carbon monoxide/nitrogen)
    Carminic acid
    Cerous nitrate
    Charcoal, activated (Norit A)
    Chloramine T ( N-chloro-4-toluenesulfonamide, sodium salt; see note)
    Chloroauric acid (gold chloride, HAuCl4xH2O)
    Chloroform
    Chromotropic acid (2,5-dihydroxynaphthalene-2,7-disulfonic acid)
    Citric acid
    Copper foil, mesh or wire*
     o-Cresol (2-methylphenol)
    Copper (II) sulfate
    Cyclohexane

    Diethyl ether
     p-Dimethylaminobenzaldehyde (4-dimethylaminobenzaldehyde)
     p-Dimethylaminocinnamaldehyde (4-dimethylaminocinnamaldehyde)
     o-Dinitrobenzene (1,2-dinitrobenzene)
    Dinitrophenol
    Diphenylamine
    Diphenylamine sulfate
    Diphenylcarbazide
    Disodium hydrogen orthophosphate
    Disodium tetraborate
    5,5'-Dithiobis(2-nitrobenzoic acid) (Ellman's reagent)
    Dithiooxamide
    Dithizone (diphenylthiocarbazone)

    Ethanol*
    Ethyl acetate

    Ferric ammonium sulfate*
    Ferric chloride
    Ferric nitrate (nonahydrate)
    Ferrous sulfate
    Fluorescein
    1-Fluoro-2,4-dinitrobenzene
    Folin-Ciocalteau reagent
    Formaldehyde*
    Formic acid*
    Furfuraldehyde

    Glycine

     n-Hexane
    Hippuric acid*
    Hydrochloric acid (concentrated, relative density 1.18)
    Hydrogen peroxide*

    Iodine
    Isopropyl acetone

    Ketodase (-glucuronidase 5000 units/ml)

    Lead acetate*
    Lithium sulfate

    Magnesium (powder)
    Manganous sulfate
    Mercuric chloride*
    Mercurous nitrate
    Metaphosphoric acid

    Methanol*
    2-Methoxyethanol (ethylene glycol monomethyl ether)
    Methyl  iso-butyl ketone (isopropylacetone)
    Methyl tertiary-butyl ether

     N-(1-Naphthyl)ethylenediamine hydrochloride
    Nicotinamide adenine dinucleotide 
    Nitric acid (concentrated, relative density 1.42)
     p-Nitrobenzaldehyde (4-nitrobenzaldehyde)
    4-( p-Nitrobenzyl)pyridine (4-(4-nitrobenzyl)pyridine)

    Orthophosphoric acid ( o-phosphoric acid, H3PO4) (850 g/kg)
    Oxalyldihydrazide (oxalylhydrazide)
    Oxalic acid*

    Paraffin wax
    Perchloric acid (700 g/kg)
    Petroleum ether (40-60C boiling fraction)
    Phenol*
    Phosphorous acid (metaphosphoric acid, H3PO3)
    Platinic chloride (platinum (IV) chloride)
    Potassium cyanide*
    Potassium dichromate
    Potassium ferricyanide (hexacyanoferrate (III))
    Potassium ferrocyanide (hexacyanoferrate (II))
    Potassium iodide
    Potassium nitrite
    Potassium permanganate
    Potassium sodium tartrate tetrahydrate (Rochelle salt)
    Potassium thiocyanate*
    Propan-2-ol*
    Pyridine

    Salicylaldehyde (2-hydroxybenzaldehyde)
    Semicarbazide hydrochloride ( N-aminourea hydrochloride)
    Silica (precipitated)
    Silver dithiocarbamate
    Silver nitrate
    Sodium acetate, anhydrous
    Sodium acetate dihydrate
    Sodium azide
    Sodium bicarbonate
    Sodium bisulfite
    Sodium bitartrate
    Sodium bromide*
    Sodium carbonate
    Sodium chlorate*
    Sodium chloride
    Sodium dihydrogen orthophosphate

    Sodium dithionite (N.B. This compound must be stored in a desiccator)
    Sodium hydrogen orthophosphate
    Sodium hydroxide
    Sodium hypochlorite*
    Sodium fluoride*
    Sodium iodate*
    Sodium iodide*
    Sodium molybdate
    Sodium nitrate*
    Sodium nitrite*
    Sodium nitroprusside
    Sodium periodate
    Sodium pyrophosphate (tetra-sodium pyrophosphate decahydrate)
    Sodium rhodizonate (rhodizonic acid, sodium salt)
    Sodium sulfate (anhydrous)
    Sodium sulfide*
    Sodium sulfite*
    Sodium tungstate
    Stannous chloride
    Starch (solid)
    Sulfanilic acid
    Sulfuric acid (concentrated, relative density 1.83)

    Tartaric acid
    Tergitol
    Tetraethylenepentamine
    Thallous sulfate*
    Thiobarbituric acid (4,6-dihydroxy-2-mercaptopyrimidine)
    Toluene
     o-Toluidine (2-methylaniline)
    Trichloroacetic acid*
    Triethylamine
    Tris(hydroxymethyl)aminomethane (free base)
    Turmeric (the spice)

    Urea

     m-Xylene (1,3-dimethylbenzene)

    Zinc (granulated)
    Zinc acetate
    Zinc phosphide*

    Annex 2

    Conversion factors for mass and molar concentrations

    The units given here are those normally used to report the results of
    measurements performed on fluids such as blood or urine.

    Analyte               Mass/molar               Molar/mass

    Acetylsalicylic acid  see salicylate ion
    Aluminium             g/l  0.0371 = mol/l   mol/l  27 = g/l
    Amitriptyline         g/l  0.00361 = mol/l  mol/l  277 = g/l
    Arsenic               mg/l  13.3 = mol/l     mol/l  0.0749 = mg/l

    Barbital              mg/l  5.43 = mol/l     mol/l  0.184 = mg/l
    Borate ion            mg/l  17.0 = mol/l     mol/l  0.0588 = mg/l
    Bromide ion           mg/l  12.52 = mol/l    mol/l  0.0799 = mg/l
    Bromoxynil            mg/l  3.61 = mol/l     mol/l  0.277 = mg/l

    Cadmium               g/l  8.90 = nmol/l     nmol/l  0.112 = g/l
    Carbamazepine         mg/l  4.24 = mol/l     mol/l  0.236 = mg/l
    Chloroquine           mg/l  3.13 = mol/l     mol/l  0.320 = mg/l
    Clomethiazole         mg/l  6.17= mol/l      mol/l  0.162 = mg/l
    Copper                mg/l  15.7 = mol/l     mol/l  0.0636 = mg/l
    Cyanide               mg/l  38.5 = mol/l     mol/l  0.026 = mg/l

    Dapsone               mg/l  4.03 = mol/l     mol/l  0.248 = mg/l
    Diazepam              mg/l  3.51 = mol/l     mol/l  0.285 = mg/l
    Digoxin               g/l  1.28 = nmol/l     nmol/l  0.781 = g/l
    Dinoseb               mg/l  4.17 = mol/l     mol/l  0.240 = mg/l
    DNOC                  mg/l  5.05 = mol/l     mol/l  0.198 = mg/l

    Ethanol               g/l  21.7 = mmol/l      mmol/l  0.046 = g/l
    Ethylene glycol       g/l  16.1 = mmol/l      mmol/l  0.062 = g/l

    Fluoride ion          mg/l  52.6 = mol/l     mol/l  0.019 = mg/l

    Hippurate ion         g/l  5.61 = mmol/l      mmol/l  0.178 = g/l

    Imipramine            g/l  0.00357 = mol/l  mol/l  280 = g/l
    Ioxynil               mg/l  2.696 = mol/l    mol/l  0.371 = mg/l
    Iron                  mg/l  17.9 = mol/l     mol/l  0.0559 = mg/l
    Isoniazid             mg/l  7.29 = mol/l     mol/l  0.137 = mg/l

    Lead                  mg/l  4.83 = mol/l     mol/l  0.207 = mg/l
    Lidocaine             mg/l  4.27 = mol/l     mol/l  0.234 = mg/l
    Lithium               mg/l  0.144 = mmol/l    mmol/l  6.94 = mg/l

    Analyte               Mass/molar               Molar/mass

    Mercury               g/l  4.99 = nmol/l     nmol/l  0.201 = g/l
    Methanol              g/l  31.3 = mmol/l      mmol/l  0.032 = g/l
    Methaqualone          mg/l  4.00 = mol/l     mol/l  0.250 = mg/l
    Morphine              mg/l  3.51 = mol/l     mol/l  0.285 = mg/l

    Nitrite ion           mg/l  21.7 = mol/l     mol/l  0.040 = mg/l
    Nortriptyline         mg/l  3.80 = mol/l     mol/l  0.263 = mg/l

    Paracetamol           mg/l  0.00661 = mmol/l  mmol/l  151= mg/l
    Paraquat ion          mg/l  5.37 = mol/l     mol/l  0.186 = mg/l
    Phenobarbital         mg/l  4.31 = mol/l     mol/l  0.232 = mg/l
    Phenprocoumon         mg/l  3.57 = mol/l     mol/l  0.280 = mg/l
    Phenytoin             mg/l  3.96 = mol/l     mol/l  0.252 = mg/l
    Primidone             mg/l  4.58 = mol/l     mol/l  0.218 = mg/l
    Propan-2-ol           g/l  16.64 = mmol/l     mmol/l  0.060 = g/l

    Quinidine             mg/l  3.08 = mol/l     mol/l  0.325 = mg/l

    Salicylate ion        mg/l  0.00729 = mmol/l  mmol/l  137 = mg/l
    Sulfite ion           mg/l  12.5 = mol/l     mol/l  0.080 = mg/l

    Thallium              mg/l  4.89 = mol/l     mol/l  0.204 = mg/l
    Theophylline          mg/l  5.55 = mol/l     mol/l  0.180 = mg/l
    Thiocyanate ion       mg/l  17.2 = mol/l     mol/l  0.058 = mg/l
    Tolbutamide           mg/l  3.70 = mol/l     mol/l  0.270 = mg/l

    Valproate ion         mg/l  6.98 = mol/l     mol/l  0.143 = mg/l

    Warfarin              mg/l  3.24 = mol/l     mol/l  0.308 = mg/l

    Zinc                  mg/l  15.3 = mol/l     mol/l  0.0654 = mg/l