Chapter 32: Analytical and Forensic Toxicology

It is impossible to consider the topic of forensic toxicology without discussing analytical toxicology in detail, as analytical toxicology has its roots in forensic applications. Therefore, it is logical to discuss these mutually dependent areas together. Analytical toxicology involves the application of the tools of analytical chemistry to the qualitative and/or quantitative estimation of chemicals that may exert effects on living organisms. Generally, the chemical that is to be measured (the analyte) is a xenobiotic that may have been altered or transformed by metabolic actions of the organism. Frequently, the specimen that is to be analyzed consists of a matrix composed of body fluids or solid tissues removed from the organism. Both the identity of the analyte and the complexity of the matrix can present formidable problems to an analytical toxicologist.

Forensic toxicology involves the use of toxicology for the purposes of the law (Cravey and Baselt, 1981). Although this broad definition includes a wide range of applications, such as regulatory toxicology and urine testing to detect drug use, by far the most common application is to identify any chemical that may serve as a causative agent in inflicting death or injury on humans, or in causing damage to property. Frequently, as a result of such unfortunate incidents, charges of liability or criminal intent are brought that must be resolved by the judicial system. At times, indirect or circumstantial evidence is presented in an attempt to prove cause and effect. However, there is no substitute for the unequivocal identification of a specific chemical substance that is demonstrated to be present in tissues from the victim at a sufficient concentration to explain the injury with a reasonable degree of scientific probability or certainty. For this reason, forensic toxicology and analytical toxicology have long shared a mutually supportive partnership.

To aid in deciding whether adverse effects of xenobiotics contribute to death, injury, or other harm to persons or property, great efforts are made to initiate and implement analytical procedures in a forensically credible manner. Laws prescribing punishment to drug-impaired drivers are evidence of attempts to mitigate impaired driving. The measurement of ethanol in blood or breath at specific concentrations is generally required to prove impairment by this agent (Fisher et al., 1968). Similarly, the decade of the 1980s saw a growing response by society to the threat of drug abuse. Identification of drug users through testing urine for the presence of drugs or their metabolites, using methods and safeguards developed by forensic toxicologists, have become required by law (Department of Health and Human Services, 1988).

The diagnosis and treatment of health problems induced by chemical substances and the closely allied field of therapeutic drug monitoring also rely greatly on analytical toxicology. Although the analytes are present in biological matrices similar to those obtained for traditional forensic toxicology, the results must be reported rapidly to be of use to clinicians in treating patients. This requirement of a rapid turnaround time often limits the number of chemicals that can be measured because analytical methods, equipment, and personnel must all be available for a swift response to toxicological emergencies. More recently, analytical toxicology methods have also been employed for the detection of drugs and other chemicals used for the purpose of performance-enhancement, which can also require rapid reporting in selected settings, including athletic events.

Occupational toxicology and regulatory toxicology also require analytical procedures for their implementation or monitoring. In occupational toxicology, the analytical methods used to monitor threshold limit values and other criteria for estimating the exposure of workers to toxic hazards may utilize simple, nonspecific, but economical screening devices. However, to determine the actual exposure of a worker, it is necessary to analyze blood, urine, breath, or another specimen by employing methods similar to those used in clinical or forensic toxicology. For regulatory purposes, a variety of matrices (eg, food, water, air) must be examined for extremely small quantities of analytes. Frequently, this requires the use of sophisticated methodology with extreme sensitivity. Both of these applications of analytical toxicology impinge on forensic toxicology because an injury or occupational disease in a worker can result in a legal proceeding, as well as can occur with a violation of a regulatory law.

Other applications of analytical toxicology occur frequently during the course of experimental studies. Confirmation of the concentration of dosing solutions and monitoring of their stability often can be accomplished with the use of simple analytical techniques. The bioavailability of a dose may vary with the route of administration and the vehicle used. Blood concentrations can be monitored as a means of establishing this important parameter. In addition, an important feature in the study of any toxic substance is the characterization of its metabolites as well as the distribution of the parent drug, together with its metabolites, to various tissues. This requires sensitive, specific, and valid analytical procedures. Similar analytical studies can be conducted within a temporal framework to gain an understanding of the dynamics of the absorption, distribution, metabolism, and excretion of toxic chemicals.

It is evident that analytical toxicology is intimately involved in many aspects of experimental and applied toxicology. Because toxic substances include all chemical types and the measurement of toxic chemicals may require the examination of biological or nonbiological matrices, the scope of analytical toxicology is broad. Nevertheless, a systematic approach and a reliance on the practical experience of generations of forensic toxicologists can be used in conjunction with the sophisticated tools of analytical chemistry to provide the data needed to better understand the hazards of toxic substances.

In light of the statement by Paracelsus five centuries ago, “All substances are poisons: there is none which is not a poison,” analytical toxicology potentially encompasses all chemical substances. Forensic toxicologists learned long ago that when the nature of a suspected poison is unknown, a systematic, standardized approach must be used to identify the presence of most common toxic substances. An approach that has stood the test of time was first suggested by Chapuis in 1873 in Elements de Toxicologie. It is based on the origin or nature of the toxic agent (Petersen et al., 1923). Such a system can be characterized as follows:

1. Gases

2. Volatile substances

3. Corrosive agents

4. Metals

5. Anions and nonmetals

6. Nonvolatile organic substances

7. Miscellaneous

Closely related to this descriptive classification is the method for separating a toxic agent from the matrix in which it is embedded. The matrix is generally a biological specimen such as a body fluid or tissue. The agent of interest may exist in the matrix in a simple solution or may be bound to protein and other cellular constituents. The challenge is to separate the toxic agent in sufficient purity and quantity to permit it to be characterized and quantified. At times, the parent compound is no longer present in large enough amounts to be separated. In this case, known metabolites may indirectly provide a measure of the parent substance (Hawks and Chiang, 1986). With other substances, interaction of the poison with tissue components may require the isolation or characterization of a protein adduct (SanGeorge and Hoberman, 1986; Stockham and Blanke, 1988). Methods for separation have long provided a great challenge to analytical toxicologists. Over the previous 20 years, improved methods have become available that permit direct measurement of some analytes without prior separation from the matrix.

Gases are most simply measured by means of gas chromatography. Some gases are extremely labile, and the specimen must be collected and preserved at temperatures as low as that of liquid nitrogen. Generally, the gas is carefully liberated by incubating the specimen at a predetermined temperature in a sealed container. The gas, freed from the matrix, collects in the empty “headspace” between the specimen and the sealed container, where it can be sampled and injected into the gas chromatograph. Other gases, such as carbon monoxide, interact with proteins. These gases can be carefully released from the protein, or the adduct can be measured independently, as in the case of carboxyhemoglobin.

Volatile substances are generally liquids of a variety of chemical types. The temperature at which they boil is sufficiently low such that older methods of separation employed microdistillation or diffusion techniques. Gas chromatography is the simplest approach for simultaneous separation and quantitation of volatile substances. The simple alcohols (eg, ethanol) can be measured by injecting a diluted body fluid directly onto the analytical column of the gas chromatograph. A more common approach is to use the headspace technique, as is done for gases, after incubating the specimen in a sealed vial at an elevated temperature.

Corrosives include mineral acids and bases. Many corrosives consist of ions that are normal tissue constituents. Clinical chemical techniques can be applied to detect these ions when they are in great excess over normal concentrations. Because these ions are endogenous constituents, the corrosive effects at the site of contact of the chemical, together with other changes in blood chemistry values, can confirm the ingestion of a corrosive substance.

Metals are encountered frequently as occupational and environmental hazards. Elegant analytical methods are available for most metals even when they are present at extremely low concentrations. Classic separation procedures involve destruction of the organic matrix by chemical or thermal oxidation. This leaves the metal to be identified and quantified in the inorganic residue. Unfortunately, this prevents the determination of the metal in the oxidation state or in combination with other elements, as it existed when the metal compound was absorbed. For example, the toxic effects of metallic mercury, mercurous ion, mercuric ion, and dimethylmercury are all different. Selected analytical methods must be capable of determining the speciation and relative amount of each form present to interpret the degree of toxicity. The analytical difficulty in doing so has resulted in the unfortunate practice of describing the toxicity of metals as if each metal existed as a single entity.

Toxic anions and nonmetals present an analytical challenge. With the exception of phosphorus, these agents are rarely encountered in an uncombined form. Some anions can be trapped in combination with a stable cation, after which the organic matrix can be destroyed, as with metals. Others can be separated from the bulk of the matrix by dialysis, after which they are detected by colorimetric or chromatographic techniques. Still others are detected and measured by ion-specific electrodes.

The nonvolatile organic substances constitute the largest group of substances that must be considered by analytical toxicologists. This group includes drugs, both prescribed and illicit, pesticides, natural products, pollutants, and industrial compounds. These substances are solids or liquids with high boiling points. Thus, separation procedures generally rely on differential extractions of biological fluids and tissues (Fig. 32-1). These extractions often are inefficient, and recovery of the analyte from the matrix may be poor. When the nature of the analyte is known, immunoassay procedures, if available are useful because they allow a toxicologist to avoid using separation procedures. These compounds can be classified as follows:

1. Organic strong acids

2. Organic weak acids

3. Organic bases

4. Organic neutral compounds

5. Organic amphoteric compounds

Separation of the classes specified above is generally achieved by adjusting the acidity of the aqueous matrix and extracting with a water-immiscible solvent or a solid-phase sorbent material. Finally, a miscellaneous category must also be included to cover the large number of compounds that cannot be detected by the routine application of the methods described above. Venoms and other toxic mixtures of proteins, or uncharacterized constituents fall into this class. Frequently, if antibodies can be produced against the active constituent, an immunoassay may be the most practical means of detecting and measuring these highly potent and difficult-to-isolate substances. Unfortunately, unless highly specific monoclonal antibodies are used, the analytical procedure may not be acceptable for forensic purposes. Most frequently, specific analytical procedures must be developed for each analyte of this type. At times, biological endpoints are utilized to semiquantify the concentration of the isolated product.

In almost all experimental studies in toxicology, an agent, generally a single chemical substance is administered in known amounts to an organism. It is universally acknowledged that the chemical under study must be either pure, or the nature of any contaminant well-characterized, to enable interpretation of the experimental results with validity. However, it is a common practice to proceed with the experimental study without verifying the identity and purity of the compound. Not only does this practice lead to errors in establishing an accurate dose, but, depending on the nature of the study, other erroneous conclusions may be drawn. For example, the presence of related compounds in the dosage form of a tricyclic antidepressant led to erroneous conclusions about the metabolic products of the drug when it was administered together with the unidentified contaminants (Saady et al., 1981). An even greater error may result when a small amount of a contaminant may be supertoxic. A well-publicized example of this error involved the presence of dioxin in mixtures of the defoliants 2,4-D and 2,4,5-T (Panel on Herbicides, 1971) used during the Vietnam War as Agent Orange. Some of the adverse effects of Agent Orange may have been due to the low concentration of dioxin in those mixtures. Other researchers have reported that the toxicity of mixtures of polybrominated biphenyls may be due to the high toxicity of specific components, whereas other brominated biphenyls are relatively nontoxic (Mills et al., 1985).

A related application of analytical toxicology is the monitoring of dosage forms or solutions for stability throughout the course of an experimental study. Chemicals may degrade when in contact with air, by exposure to ultraviolet or other radiation, by interaction with constituents of the vehicle or dosing solution, and by other means. Developing an analytical procedure by which these changes can be recognized and corrected is essential in achieving consistent and reliable results over the course of a study (Blanke, 1987; Peters et al., 2007).

Finally, analytical methods are necessary to determine the bioavailability of a compound that is under study. Some substances with low water solubility are difficult to introduce into an animal, and a variety of vehicles may be investigated. However, a comparison of the blood concentrations for the compound under study provides a simple means of comparing the effectiveness of vehicles. Introducing a compound into the stomach in an oil vehicle may not be the most effective means of enhancing the absorption of that compound (Granger et al., 1987). Rather than observing dose–effect relationships, it may be more accurate to describe blood (serum) concentration–effect relationships.

The duties of a forensic toxicologist in postmortem investigations include the qualitative and quantitative analysis of drugs or poisons in biological specimens collected at autopsy and the interpretation of the analytical findings with respect to the physiological and behavioral effects of the detected chemicals on the deceased at the time of injury and/or death.

The complete investigation of the cause or causes of sudden death is an important civic and legal responsibility. The responsibility of establishing the cause of death rests with the medical examiner or coroner, but success in arriving at the correct conclusion often depends on the combined efforts of the pathologist and the toxicologist. The cause of death in cases of poisoning cannot be proved beyond contention without toxicological analysis that confirms the presence of the toxicant in either body fluids or tissues of the deceased.

Many drugs or poisons do not produce characteristic pathological lesions; their presence in the body can be demonstrated only by chemical methods of isolation and identification. If toxicological analyses are limited, deaths resulting from poisoning may be erroneously ascribed to an entirely different cause or poisoning may be designated as the cause of death without empirical proof. Thus, limiting analysis may have significant legal and social consequences.

Additionally, a toxicologist can furnish valuable evidence concerning the circumstances surrounding a death. Such cases commonly involve demonstrating the presence of intoxicating concentrations of ethanol in victims of automotive or industrial accidents, or measurements of concentrations of carbon monoxide in fire victims. Arson is commonly used to conceal homicide. The degree of carbon monoxide saturation of the blood may indicate whether the deceased died as a result of the fire or was dead before the fire started. Also, licit or illicit psychoactive drugs often play a significant role in the circumstances associated with sudden or violent death. The behavioral toxicity of many illicit drugs may explain the bizarre or “risk-taking” behavior of the deceased that led to his or her demise. At times, a negative toxicological finding is of particular importance in assessing the cause of death. For example, toxicology studies may demonstrate that a person with a seizure disorder was not taking the prescribed medication and that this noncompliance contributed to the fatal event.

Additionally, the results of postmortem toxicological testing provide valuable epidemiological and statistical data. Forensic toxicologists are often among the first to alert the medical community to new epidemics of substance abuse (Poklis, 1982) and the dangers of abusing over-the-counter drugs (Garriott et al., 1985). Similarly, they often determine the chemical identity and toxicity of novel analogs of psychoactive agents that are subject to abuse, including “designer drugs” such as “china white” (methylfentanyl) (Henderson, 1988), “ecstasy” (methylenedioxy-methamphetamine) (Dowling et al., 1987), and GHB (gamma-hydroxybutyric acid) (Andresen et al., 2011). More recently, several newer groups of designer drugs, including the synthetic cathinones and derivatives such as 4-methylmethcathinone (mephedrone) and methylone (Cawrse et al., 2012; Prosser and Nelson, 2012), 3,4-methylenedioxypyrovalerone and 5,6-methylenedioxy-2-aminoindane (MDAI) (Gallagher et al., 2012; Coppola and Mondola, 2012; Ross et al., 2012), and products containing synthetic cannabinoids, such as “K2” and “Spice” (Seely et al., 2012), among others, have emerged as new trends, particularly among young age groups (Rosenbaum et al., 2012).

Today, there are numerous specialized areas of study in the field of toxicology; however, it is the forensic toxicologist who is obliged to assist in the determination of the cause of death for a court of law and who has been historically recognized by the title “toxicologist.”

Until the 19th century, physicians, lawyers, and law enforcement officials harbored extremely faulty notions about the signs and symptoms of poisoning (Thorwald, 1965). Unless a poisoner was literally caught in the act of the crime, there was no way to establish whether the victim died from poisoning. In the early 18th century, a Dutch physician, Hermann Boerhoave, theorized that various poisons in a hot, vaporous condition yield characteristic odors. He placed substances suspected of containing poisons on hot coals and evaluated their smells. Although Boerhoave was not successful in applying his method, he was the first to suggest a chemical method for proving the presence of poison.

White arsenic (arsenic trioxide) has been widely used with murderous intent for over a thousand years. Therefore, it is not surprising that the first milestones in the chemical isolation and identification of a poison in body tissues and fluids were described for arsenic. In 1775, Karl Wilhelm Scheele, a Swedish chemist, discovered that white arsenic is converted to arsenous acid by chlorine water. The addition of metallic zinc reduced the arsenous acid to poisonous arsine gas. If gently heated, the evolving gas would deposit metallic arsenic on the surface of a cold vessel. In 1821, Serullas utilized the decomposition of arsine for the detection of small quantities of arsenic in stomach contents and urine in poisoning cases. In 1836, James M. Marsh, a chemist at the Royal British Arsenal in Woolwich, applied Serullas’ observations in developing the first reliable method to determine the presence of an absorbed poison in body tissues and fluids such as liver, kidney, and blood. After acid digestion of the tissues, Marsh generated arsine gas, which was drawn through a heated capillary tube. The arsine decomposed, leaving a dark deposit of metallic arsenic. Quantitative measures were performed by comparing the length of the deposit from known concentrations of arsenic with those of the test specimens.

The 1800s witnessed the development of forensic toxicology as a scientific discipline. In 1814, Mathieiv J. B. Orfila (1787–1853), widely considered the “father of toxicology,” published Traité des Poisons, the first systematic approach to the study of the chemical and physiological nature of poisons (Gettler, 1977). Orfila’s role as an expert witness in many famous murder trials, particularly his application of the Marsh test for arsenic in the trial of the poisoner Marie Lafarge, aroused both popular and scholarly interest in the new science. As dean of the medical faculty at the University of Paris, Orfila trained numerous students in forensic toxicology.

The first successful isolation of an alkaloidal poison was performed in 1850 by Jean Servias Stas, a Belgian chemist, using a solution of acetic acid in warm ethanol to extract nicotine from the tissues of the murdered Gustave Fougnie. As modified by the German chemist Fredrick Otto, the Stas–Otto method was quickly applied to the isolation of numerous alkaloidal poisons, including colchicine, coniine, morphine, narcotine, and strychnine. In the latter half of the 19th century, European toxicologists were in the forefront of the development and application of forensic sciences, providing valuable evidence of poisoning. A number of these trials became “causes célèbres” and the testimony of forensic toxicologists captured the imagination of the public and increased awareness of the development and application of toxicology. It was thought that murderers could no longer poison with impunity.

In the United States, Rudolph A. Witthaus, professor of chemistry at Cornell University Medical School, made many contributions to toxicology and called attention to the new science by performing analyses for the city of New York in several famous morphine poisoning cases, including the murder of Helen Potts by Carlyle Harris and that of Annie Sutherland by Dr Robert W. Buchanan. In 1911, Witthaus and Tracy C. Becker edited a four-volume work on medical jurisprudence, forensic medicine, and toxicology—the first standard forensic textbook published in the United States. In 1918, the city of New York established a medical examiner’s system, and the appointment of Dr Alexander O. Gettler as toxicologist marked the beginning of modern forensic toxicology in this country. Although Dr Gettler made numerous contributions to the science, perhaps his greatest was the training and direction he gave to future leaders in forensic toxicology. Many of his associates went on to direct laboratories in medical examiner and coroner systems in major urban centers throughout the country.

In 1949, the American Academy of Forensic Sciences (AAFS) was established to support and further the practice of legal (forensic) medicine in the United States. The members of the Toxicology Section represented the vast majority of forensic toxicologists working in medical examiners’ and coroner offices’, as well as numerous referral laboratories. Several other international, national, and local forensic science organizations, such as the Society of Forensic Toxicologists and the California Association of Toxicologists, offer a forum for the exchange of scientific data pertaining to analytical techniques and case reports involving new or infrequently used drugs and poisons. The International Association of Forensic Toxicologists (TIAFT) founded in 1963 with over 1400 members from all regions of the world facilitates worldwide collaboration in resolving the technical problems confronting toxicology.

In 1975, the American Board of Forensic Toxicology (ABFT) was formed to examine and certify forensic toxicologists. One of the stated objectives of the board is “to make available to the judicial system, and other publics, a practical and equitable system for readily identifying those persons professing to be specialists in forensic toxicology who possess the requisite qualifications and competence.” Those certified as Diplomates of the Board must have a doctor of philosophy or doctor of science degree, have at least three years of full-time professional experience, and pass a written examination. In 2000, the Board began certifying “forensic toxicology specialists.” Specialists must have a master’s or bachelor’s degree and three years of full-time professional experience and must pass a written examination. In 2012, there are approximately 140 Diplomates and 95 Specialists certified by the Board. In 1998, the Board began an accreditation program for forensic toxicology laboratories. ABFT accredited laboratories must conform to standards of having qualified, experienced personnel and forensically sound procedures for the handling of evidence, analysis of specimens, and reporting of results. Laboratories must pass bi-annual on-site inspections, which include a review of laboratory procedures and casework.

The toxicological investigation of a poison death may be divided into three steps: (1) obtaining the case history and suitable specimens, (2) the toxicological analyses, and (3) the interpretation of the analytical findings.

Case History and Specimens

Today, thousands of compounds are readily available that are lethal if ingested, injected, or inhaled. Usually, a limited amount of specimen is available on which to perform analyses; therefore it is imperative that, before the analyses are initiated, as much information as possible concerning the facts of the case be collected. The age, sex, weight, medical history, and occupation of the decedent as well as any treatment administered before death, the gross autopsy findings, the drugs available to the decedent, and the interval between the onset of symptoms and death should be noted. In a typical year, a postmortem toxicology laboratory will perform analyses for such diverse poisons as over-the-counter medications (eg, analgesics, antihistamines), prescription drugs (eg, benzodiazepines, opioids), drugs of abuse (eg, cocaine, marijuana, methamphetamine), and gases (eg, inhalants, carbon monoxide). Obviously, a thorough investigation of the death scene including a tentative identification of the administered poison is helpful prior to beginning the analysis (Ernst et al., 1982).

The pathologist at autopsy usually performs the collection of postmortem specimens for analysis. Specimens of many different body fluids and organs are necessary, as drugs and poisons display varying affinities for body tissues (Fig. 32-2). Therefore, detection of a poison is more likely in a tissue in which it accumulates. A large quantity of each specimen is needed for thorough toxicological analysis because a procedure that extracts and identifies one compound or class of compounds may be ineffective in extracting and identifying others (Table 32-1). Autolytic and putrefactive changes, however, may reduce specimen quality and therefore alter the selection and utility of individual specimens on a case-by-case basis. Further improvements in our knowledge regarding degradation mechanisms, as well as specimen-handling protocols to increase storage stability, may enable the forensic toxicologist to circumvent possible analytical and interpretive difficulties (Dinis-Oliveira et al., 2010).

When collecting the specimens, the pathologist labels each container with the date and time of autopsy, the name of the decedent, the identity of the sample, an appropriate case identification number, and his or her signature or initials. It is paramount that the handling of all specimens be authenticated and documented. A form developed at the collection site that identifies each specimen is submitted to the laboratory with the specimens. The form is signed and dated by the pathologist, and subsequently by any individual handling, transferring, or transporting the specimens. In legal terms, this form constitutes a “chain-of-custody” of specimens documenting all transfers. The chain-of-custody enables a toxicologist to introduce his or her results into legal proceedings, having established that the specimens analyzed were obtained from the decedent.

Fluids and tissues should be collected before embalming, as this process will dilute or chemically alter the poisons present, rendering their detection difficult or impossible. Conversely, methyl or ethyl alcohol may be a constituent of embalming fluid, thereby affecting interpretation of the analytical findings.

Although forensic toxicology laboratories typically receive blood, urine, liver tissue, and/or stomach contents for identification of xenobiotics, they have been increasingly called upon to meet the analytical challenges of many alternative types of samples (Skopp, 2004; Flanagan et al., 2005; Kronstrand and Druid, 2006; Gallardo and Queiroz, 2008). Nontraditional matrices, such as bone marrow, hair, and nails, among others, may be submitted to the laboratory. For example, on occasion, toxicological analysis is requested for cases of burned, exhumed, putrefied, and skeletal remains. In such instances, it is necessary to analyze unusual specimens such as bone marrow, hair, nails, skeletal muscle, vitreous humor, and even insects (Inoue, 1992). Numerous drugs have been successfully identified in bone marrow and bone washings from skeletal remains even after decomposition and burial (Benko, 1985; Drummer, 2010; Cartiser et al., 2011). Similarly, the vitreous humor of the eye is isolated and sequestered from putrefaction, charring, and trauma; thus, it is a useful specimen for the detection of most drugs, anions, and even volatile poisons such as alcohols, ketones, and glycols (Coe, 1993; Bévalot et al., 2011). Hair analysis is a rapidly growing technique in forensic toxicology and has been used to measure individual exposure to heavy metals, such as arsenic, mercury, and lead, as well as many drugs of abuse and other pharmaceuticals, pesticides, and plastics (Yamaguichi et al., 1975; McKenzie, 1978; Baumgartner et al., 1981; Hambidge, 1973; Puschel et al., 1983; Suzuki, 1984; Marigo et al., 1986; Schroeder and Nason, 1989; Strang et al., 1990; Martz, 1988; Harkey and Henderson, 1989; Shen et al., 2002; Boumba et al., 2006). Analysis of hair for selected drugs has been utilized as an adjunct specimen in forensic settings for postmortem examinations for many years (Baumgartner et al., 1979; Couper et al., 1995; Gaillard and Pepin, 1997; Pragst et al., 1997; Rollins et al., 1997; Yegles et al., 1997; Villain et al., 2005; Muller et al., 2000; Pragst and Balikova, 2006; Kintz, 2010; Barroso et al., 2011). Nails, another keratinized matrix, have also been used to determine exposure to selected xenobiotics in both antemortem and postmortem cases (Palmeri et al., 2000; Garside, 2010). Limited data are available to support a direct correlation between quantitative hair and nail values and drug doses in forensic cases; however, qualitative results have been accepted as indicators of previous drug or xenobiotic exposure.

Finally, in severely decomposed bodies, the absence of blood and/or the scarcity of solid tissues suitable for analysis have led to the collection and testing of maggots (fly larvae) feeding on the body (Pounder, 1991). The fundamental premise underlying maggot analysis is that if drugs or intoxicants are detected, they could only have originated from the decedent’s tissues on which the larvae were feeding. Surprisingly, analysis of maggots is rather straightforward, requiring no special methodology beyond that routinely applied in toxicology laboratories. Case reports have documented the detection of numerous drugs and intoxicants in maggots collected from decomposed bodies. The compounds detected include barbiturates, benzodiazepines, phenothiazines, morphine, and malathion. Controlled studies in which maggots were allowed to feed on tissues to which drugs had been added have demonstrated the accumulation of propoxyphene, amitriptyline and nordiazepam, among others, in the larvae (Goff et al., 1993; Gagliano-Candela and Aventaggiato, 2001; Pien et al., 2004).

Toxicological Analysis

Before the analysis begins, several factors must be considered, including the amount of specimen available, the nature of the poison sought, and the possible biotransformation of the poison. In cases involving oral administration of the poison, the gastrointestinal (GI) contents are analyzed first because large amounts of residual unabsorbed poison may be present. The urine may be analyzed next, as the kidney is the major organ of excretion for most poisons and high concentrations of toxicants and/or their metabolites often are present in urine. After absorption from the GI tract, drugs or poisons are carried to the liver before entering the general systemic circulation; therefore, the first analysis of an internal organ is conducted on the liver. If a specific poison is suspected to have caused or contributed to a death, the toxicologist may first analyze the tissues and fluids in which the poison concentrates.

A thorough knowledge of drug biotransformation is often essential before an analysis is performed. The parent compound and any major pharmacologically-active metabolites should be isolated and identified. In some instances, the metabolites provide the only evidence that a drug or poison has been administered. Many screening tests, such as immunoassays, are specifically designed to detect not the parent drug but its major urinary metabolite. An example of the relationship of pharmacokinetic and analytical factors is provided by cocaine. The major metabolites of cocaine biotransformation are benzoylecgonine and ecgonine methyl ester (Fig. 32-3). The co-ingestion of alcohol with cocaine results in the hepatic transesterification of cocaine to form cocaethylene (Hime et al., 1991), a psychoactive metabolite. The disposition of these compounds in various body fluids and hair is shown in Fig. 32-4. Thus, the initial testing of urine to determine cocaine use is performed with immunoassays specifically designed to detect the presence of benzoylecgonine, the major urinary metabolite. In contrast, if saliva or hair is tested, parent cocaine, as well as metabolites, are also sought. To determine a profile of each analyte present in a specimen, chromatographic procedures such as gas chromatography-mass spectrometry (GC-MS) are used, which facilitates the simultaneous separation and quantification of each compound.

The analysis may be complicated by the normal chemical changes that occur during the decomposition of a cadaver. The autopsy and toxicological analysis should be started as soon after death as possible. The natural enzymatic and nonenzymatic processes of decomposition and microbial metabolism may destroy a poison that was present at death or produce substances or compounds with chemical and physical properties similar to those of commonly encountered poisons. As early as the 1870s, the so-called cadaveric alkaloids isolated from the organs of putrefied bodies were known to produce color test reactions similar to those produced by morphine and other drugs. These cadaveric alkaloids resulted from the bacterial decarboxylation of the amino acids ornithine and lysine, producing putrescine and cadaverine, respectively (Evans, 1963). Similarly, during decomposition, phenylalanine is converted to phenylethylamine, which has chemical and physical properties very similar to those of amphetamine. The hydrolysis, oxidation, or reduction of proteins, nucleic acids, and lipids may generate numerous compounds, such as hydroxylated aliphatic and aromatic carboxylic acids, pyridine and piperidine derivatives, and aromatic heterocyclics such as tryptamine and norharmane (Kaempe, 1969). All these substances may interfere with the isolation, identification, and quantitation of the toxicants being sought. The concentration of cyanide and ethyl alcohol and the carbon monoxide saturation of the blood may be decreased or increased, depending on the degree of putrefaction and microbial activity. However, many poisons—such as arsenic, barbiturates, mercury, and strychnine—are extremely stable and may be detectable many years after death.

Before analysis, the purity of all chemicals used in laboratory procedures should be established. The purity of the primary reference material used to prepare calibrators and controls should be verified, and the salt form or degree of hydration should be determined (Blanke, 1989). All reagents and solvents should be of the highest grade possible and should be free of contaminants that may interfere with or distort analytical findings. For example, the chloroform contaminants phosgene and ethyl chloroformate may react with primary or secondary amine drugs to form carbamyl chloride and ethyl carbamate derivatives (Cone et al., 1982). Specimen containers, lids, and stoppers should be free of contaminants such as plasticizers, which often interfere with chromatographic or GC-MS determinations. Care should be exercised to ensure a clean laboratory environment. This is of particular concern in the analysis of metals, as aluminum, arsenic, lead, and mercury are ubiquitous environmental and reagent contaminants.

Forensic toxicology laboratories analyze specimens by using a variety of analytical procedures. Initially, nonspecific tests designed to determine the presence or absence of a class or group of analytes may be performed directly on the specimens. Examples of tests used to rapidly screen urine are the FPN (ferric chloride, perchloric, and nitric acid) color test for phenothiazine drugs and immunoassays for the detection of amphetamines, benzodiazepines, and opiate derivatives, among others. Positive results obtained with these tests must be confirmed by a second analytical procedure that identifies the particular drug. The detection limit of the confirmatory test should be lower than that of the initial nonspecific test. Some analytical procedures identify specific compounds. Even in such instances, a second test should be performed to identify and confirm the presence of the analyte. The second test should be based on a chemical or physical principle different from that of the first test. Such additional testing is performed to establish an unequivocal identification of the drugs or poisons present. Whenever possible, the most specific test for the compound of interest should be performed. Today, GC-MS and liquid chromatography-mass spectrometry (LC-MS) are the most widely applied methodology in toxicology and are generally accepted as unequivocal identification for all drugs. Analyte identification is typically based on the retention time in the chromatographic system coupled with the characteristic ion fragmentation spectrum in the mass spectrometer. The analyte mass spectrum is the pattern of mass-to-charge ion fragments and their relative abundance.

Analytical methods must be of sufficient rigor to provide accurate and reliable qualitative and quantitative data. Numerous approaches for the use of quality control have been suggested, and professional organizations have developed recommendations for the implementation of quality control in forensic laboratories (Goldberger et al., 1997; Ferrara et al., 1998; Scientific Working Group for Forensic Toxicology). The limit of detection, the lowest concentration of analyte reliably identified by the assay, and the specificity of all qualitative methods should be well documented. The laboratory must demonstrate that the assay response of blank or negative calibrators (controls) does not overlap with the response of the lowest-positive calibrator. In certain instances, qualitative identification of a poison or drug is sufficient to resolve forensic toxicology issues. However, most cases require reliable estimates of poison concentrations for forensic interpretation. For quantitative analysis, the accuracy, precision, linearity, and specificity of the procedure must also be established. Linearity should be determined by using at least one drug-free and three drug-fortified calibrators whose concentrations bracket the anticipated concentrations in the biological specimen. Precision, which statistically demonstrates the variance in the value obtained, is determined by replicate analyses of a specimen of a known concentration. Additional assay parameters, such as analyte stability and recovery from the biological matrix, for example, can also be determined. For a variety of reasons, a quantitative result occasionally will deviate spuriously from the true value. Therefore, replicate quantitative determinations are highly recommended when sufficient specimen volume is available (Blanke, 1987).

When unusual samples such as bone marrow, fingernails, hair, and maggots are analyzed, the extraction efficiency of a procedure may vary greatly, depending on the complexity of the matrix. Therefore, all calibrators and controls should be prepared in the same matrix type as the specimens and analyzed concurrently with the specimens. Often the matrix is “unique” or impossible to match, such as decomposed or embalmed tissue. In these instances, the method of “standard additions” may be used. Known amounts of the analyte of interest are added to specimen aliquots and these are analyzed. The concentration of poison in the test specimen is determined by comparing the proportional response of the “poison fortified” specimens to that of the test specimens.

Interpretation of Analytical Results

Once the analysis of the specimens is complete, the toxicologist must interpret his or her findings with regard to the physiological or behavioral effects of the toxicants on the decedent at the concentrations found. Specific questions may be answered, such as the route of administration, the dose administered, and whether the concentration of the toxicant present was sufficient to cause death or alter the decedent’s actions enough to cause his or her death. Assessing the physiological or behavioral meanings of analytical results is often the most challenging aspect confronted by the forensic toxicologist.

In determining the route of administration, the toxicologist notes the results of the analysis of the various specimens. As a general rule, the highest concentrations of a poison are found at the site of administration. Therefore, the presence of large amounts of drugs and/or poisons in the GI tract and liver indicates oral ingestion, while higher concentrations in the lungs than in other visceral organs can indicate inhalation or intravenous injection. The ratio or relative distribution of drugs in different tissues may also differentiate oral from parenteral administration (Fig. 32-5). Drugs may also be detected in the tissue surrounding an injection site following intramuscular or intravenous injection. Smoking is a popular route of administration for abusers of controlled substances (illicit) such as cocaine, heroin, marijuana, and phencyclidine. Pyrolysis of these drugs leads to the inhalation not only of the parent drug, but also of characteristic breakdown products of combustion. For example, a major pyrolysis product of “crack” cocaine smoking is anhydroecgonine methyl ester (Martin et al., 1989) (Fig. 32-3). Thus, identification of relatively high concentrations of this compound along with cocaine or cocaine metabolites in urine or other body fluids or tissues may indicate smoking as the route of cocaine administration (Jacob et al., 1990).

The presence of a toxic material in the GI tract, regardless of the quantity, does not provide sufficient evidence to establish that agent as the cause of death. It is necessary to demonstrate that absorption of the toxicant has occurred and that it has been transported by the general circulation to the target organ in order to exert its lethal effect. This is established by blood and tissue analysis. An exception to the rule is provided by strong, corrosive chemicals such as sulfuric acid, lye, and phenol, which exert their deleterious effects by directly digesting tissue, causing hemorrhage and shock. The results of urinalysis are often of little benefit in determining the physiological effects of a toxic agent. Urine results establish only that the poison was present in the body at some time before death. Correlation of urine values with physiological effects is poor because of various factors that influence the rate of excretion of specific compounds and the urine volume.

The physiological effects of most drugs and poisons are generally correlated with their concentrations in blood or blood fractions such as plasma and serum. Indeed, in living persons, this association is the basis of therapeutic drug monitoring. However, postmortem blood has been described as a fluid resembling blood that is obtained from the vasculature after death. Therefore, interpretation of postmortem blood results requires careful consideration of the case history, the site of collection, and postmortem changes. The survival time between the administration of a poison and death may be sufficiently long to permit biotransformation and excretion of the agent. Blood values may appear to be nontoxic or consistent with therapeutic administration. Death from hepatic failure after an acetaminophen overdose usually occurs at least three to four days after ingestion. Postmortem acetaminophen concentrations in blood may be consistent with the ingestion of therapeutic doses. Therefore, fatal acetaminophen overdose is determined by case history, central lobular necrosis of the liver, and, if available, analysis of serum specimens collected from the decedent when he or she was admitted to the emergency department (Price et al., 1991). Furthermore, emergency medical treatment—such as the administration of fluids, plasma extenders, bicarbonate, diuretics, and blood transfusions—may dilute, remove, or enhance the elimination of toxic agents. Similarly, prolonged survival on a mechanical respirator, hemodialysis, or hemoperfusion may substantially reduce initially lethal blood concentrations of poisons.

For a long time, it was generally assumed that postmortem blood drug concentrations were more or less uniform throughout the body. However, in the 1970s, several investigators noted that postmortem concentrations of digoxin in heart blood greatly exceeded those in simultaneously collected femoral blood. They also observed that postmortem blood concentrations, particularly in heart blood, exceeded the expected values at the time of death (Vorpahl and Coe, 1978; Aderjan et al., 1979). This postmortem increase in blood digoxin concentrations was apparently due to release of the drug from tissue stores, particularly the myocardium. Subsequently, other researchers demonstrated that for many drugs, blood concentrations in the same body vary greatly depending on the site from which the specimen is collected—subclavian vein, thoracic aorta, inferior vena cava, femoral vein, and so forth. For example, in a case of fatal multiple drug ingestion, analysis of postmortem blood collected from 10 different sites demonstrated imipramine concentrations that differed by as much as 760% (2.1–16.0 mg/L) (Jones and Pounder, 1987). In an extensive investigation, Prouty and Anderson (1990) demonstrated that postmortem blood drug concentrations were not only site-dependent but also increased greatly over the interval between death and specimen collection, particularly in heart blood. This increase over the postmortem interval was most pronounced for basic drugs with large apparent volumes of distribution, such as tricyclic antidepressants.

In an overt drug overdose, postmortem blood concentrations are elevated sufficiently to render an unmistakable interpretation of fatal intoxication. However, in many cases, the postmortem redistribution of drugs may significantly affect the interpretation of analytical findings. For drugs whose volume of distribution, plasma half-life, and renal clearance vary widely from person to person or that undergo postmortem redistribution, tissue concentrations readily distinguish therapeutic administration from drug overdose (Apple, 1989). Therefore, to provide a foundation of reasonable medical certainty in regard to the role of a drug in the death of an individual, it is recommended that, in addition to heart blood, a peripheral blood specimen and tissues be analyzed.

The analysis of tissue specimens is important for the estimation of a “minimal administered dose” or body burden of a drug or poison. In order to calculate a minimum body burden, it is necessary to analyze as many different body tissues and fluids as possible to determine the concentrations of the drug present. The concentration of drug in each separate specimen is then multiplied by the total weight or volume of that particular tissue or fluid. In this manner, the total amount of drug in each different tissue or fluid is determined. The amounts of drug in each separate tissue and fluid are then added together to give the total body burden or minimal administered dose. This simple approach has often proven extremely effective in resolving legal medical issues. For example, lidocaine is commonly administered in 50- to 100-mg bolus injections as an antiarrythmic agent for ventricular arrhythmia during resuscitation efforts. Because of poor circulation and tissue perfusion during arrhythmias, lidocaine is not well distributed in the body of the victim of a fatal heart attack. Postmortem bloods often exceed 50 mg/L in such cases, whereas values for effective antiarrhythmic prophylaxis do not exceed 5 mg/L. Therefore, a blood lidocaine value of 50 mg/L may be an artifact of resuscitation efforts or might represent a fatal overdose. Tissue distribution studies have resolved this issue in both accidental and homicidal poisoning with lidocaine (Poklis et al., 1984).

Postmortem toxicology results are often used to corroborate investigative findings. Improved methods for determining drug exposure, with respect to longer time frames, are advantageous because drug concentrations in plasma, urine, and liver often reflect only the dosage taken within (at most) the last several days prior to sampling. Compared to traditional biological matrices, keratinized tissues such as hair and nails provide a longer window of detection for drugs and other compounds due to their slow growth and possible permanent retention of drugs. The presence of certain xenobiotics and their metabolites, have been demonstrated in human hair or nails at times when plasma and urine drug concentrations are not measurable (Rollins et al., 1997; Garside, 2010), which can provide evidence of exposure to these agents weeks, months, or even years prior to death. It has been suggested that for some xenobiotics, hair also serves to characterize the drug exposure over time. For example, the analysis of sequential sections of hair provides a reliable correlation with the pattern of arsenic exposure (Smith, 1964). Significant increases in the arsenic content of the root and the first 5 mm of the hair occur within hours after the ingestion of arsenic. The germinal cells are in relatively close equilibrium with circulating arsenic; thus, as arsenic concentrations in blood rise or fall, so does arsenic deposition in growing hair. Normal arsenic content in hair varies with nutritional, environmental, and physiological factors; however, the maximum upper limit of normal deposition with a 99% confidence limit in persons not exposed to arsenic is 5 mg/kg (Shapiro, 1967). Hair grows at a rate of approximately 1.06 cm per month (LeBeau et al, 2011). Therefore, analysis of 1.0-cm segments provides a monthly pattern of exposure (Fig. 32-6). Such analyses can be performed in cases of homicidal poisoning to demonstrate that increases in arsenic deposition in the victim’s hair correlate with times when a poisoner had an opportunity to administer the poison. Continuously elevated hair arsenic values indicate chronic rather than acute poisoning as the cause of death. A similar approach using segmental analysis of hair has been used to determine patterns of exposure for various drugs and their metabolites; however, care must be taken in order to account for the potential effects of haircuts, chemical treatments, and other environmental insults that may have occurred to the hair during the time frame of interest.

A new extension of forensic toxicology is the analysis of impurities of illicit drug synthesis in biological specimens. Many drugs of abuse, particularly methamphetamine are illicitly manufactured in clandestine laboratories. There are several popular methods of methamphetamine synthesis; when these are applied in clandestine laboratories, side reactions or incomplete conversion of the reactants yield an impure mixture of methamphetamine and synthetic impurities. These impurities can be characteristic of a particular synthetic method and their detection in biological specimens can indicate the use of an illicitly produced drug that is not a legal pharmaceutical product; suggest the synthetic method that was used to produce the drug; point to a possible common source of illicit production; and provide a link between manufacturers, dealers, and users. An example of impurity analysis was the detection of metabolites of α-benzyl-N-methylphenethylamine (BNMPA) in the urine of abusers of methamphetamine (Moore et al., 1996b) and in a case of fatal drug overdose involving methamphetamine (Moore et al., 1996a). BNMPA is an impurity arising from the synthesis of methamphetamine via the Leukart reaction using phenyl-2-propanone (P2P) synthesized from phenylacetic acid. Clandestine laboratories often must synthesize the P2P starting material, as its sale and distribution is regulated by the Federal Drug Enforcement Agency.

As discussed above, a wide range of tissues are available for the purpose of identifying xenobiotics in postmortem specimens. In contrast, for many years, traditional specimens used for the detection and quantification of substances in antemortem biological specimens have been limited to whole blood, plasma, serum, and urine, largely due to their ease of collection and accessibility in the living. More recently, the advent of improved analytical techniques with greater sensitivity and selectivity, including GC-MS/MS and LC-MS/MS, have expanded the array of biological specimens in which substances can be detected. These specimens include oral fluid (saliva), sweat, meconium, amniotic fluid, breast milk, and semen among others. In oral fluid, for example, the pharmacokinetics of many drugs and metabolites is closely aligned to that of blood pharmacokinetics, and thus can serve as an alternative matrix for illicit and therapeutic drug detection (Bosker and Huestis, 2009; Spiehler and Cooper, 2010), as well as detection of exposure to environmental toxicants and pesticides (Lanaro et al., 2011; Yang et al., 2011).

Over the past few decades, forensic toxicologists have become more involved in the analysis of specimens obtained from living victims of criminal poisonings. Generally, this increase in testing is a result of two types of cases: (1) administration of drugs to incapacitate victims of kidnapping, robbery, or sexual assault and (2) poisoning as a form of child abuse.

For centuries, those severely intoxicated from alcohol often became victims of kidnapping, robbery, or sexual assault. The kidnapping of drunks in seaports was a common way of obtaining sailors for long commercial voyages such as those involved in whaling. Late in the 19th century, the mixing of the powerful hypnotic chloral hydrate with alcohol produced the legendary “Mickey Finn.” While alcohol is still often a primary factor in cases of alleged sexual assault, common drugs of abuse or other psychoactive drugs are often involved (Table 32-2). Of particular concern are the many potent inductive agents medically administered prior to general anesthesia. Many of these drugs, such as benzodiazepines and phenothiazines, are available today through illicit sources or legal purchase in foreign countries. When administered surreptitiously, they cause sedation and incapacitate the victim while also producing amnesia in the victim as to the events while drugged, without causing severe central nervous system depression. These cases often present a difficult analytical challenge to the toxicologist. Usually, the victim does not bring forth an allegation of assault until 24 hours to several days after the attack. Thus, the intoxicating drug may have been largely eliminated or extensively metabolized such that extremely low concentrations of drug or metabolites are present in the victim’s blood, urine, and/or hair specimens. Sophisticated, highly sensitive analytical methods such as GC-MS or LC-MS may be required to accurately identify drugs in such specimens. To provide guidance in the choice of analytical approaches to such cases, recommendations for the toxicological investigation of sexual assaults have been formulated (LeBeau et al., 1999; Society of Forensic Toxicologists).

Poisoning as a form of child abuse involves the deliberate administration of toxic or injurious substances to a child, usually by a parent or other caregiver. The victims of such poisonings range in age from a few months to the teens. Common agents used to intentionally poison children have included syrup of ipecac, table salt, laxatives, diuretics, antidepressants, sedative-hypnotics, and narcotics (Yin, 2010; American Association of Poison Control Centers, 2011; Oral et al., 2011). The motivation for such heinous behavior is in the province of psychiatry, not toxicology. However, toxicologists must have some understanding of the nature of these poisonings to aid in the investigation of such cases. In some instances, it is a form of child battering committed by persons with low tolerance to the child’s upsetting behaviors. The poison may be given to an infant to stop its crying or be force-fed to older children as a form of punishment. Such behavior is characteristic of persons with a cultural background where violence toward children is common or who have severe personality disorders or other mental illnesses. “Munchausen syndrome by proxy” is another form of child poisoning (Murray, 1997). The term Munchausen syndrome (MS) is used to describe patients who seek admission to hospitals with apparent illness along with plausible, dramatic, albeit fictitious medical histories. Such people seek medical treatment solely to assume the role of a patient and receive the attention derived from this deception. “MS by proxy” refers to the situation in which an individual, usually a parent, presents not himself or herself but a child with a fictitious illness often induced by physical or chemical means. The purpose of the poisoning is not to kill the child but to induce signs and symptoms of illness that will assure medical attention. Thereby, the parent as caregiver gains the craved attention. Given a fictitious case history and the obvious illness of the child, these cases are almost always and understandably misdiagnosed. Often, the child may be chronically poisoned at home and in the hospital for as long as a year before suspicion leads to the collection of specimens for extensive toxicological testing. Although the parent may not have intended such an outcome, some children have died from fatal poisoning in these situations. As in the case of sexual assault, sophisticated MS testing methods may be required to detect such agents as emetine and cephaeline, the emetic alkaloids in syrup of ipecac. Testing in these cases is best performed in a laboratory with forensic experience, as positive drug findings will usually result in some form of legal proceeding.

Concerns regarding the potentially adverse consequences of substance abuse for the individual, the workplace, and society have led to widespread urine analysis for controlled or illicit drugs (Gust and Walsh, 1989). Currently, such testing is conducted routinely by the military services, regulated transportation and nuclear industries, many federal and state agencies, public utilities, federal and state criminal justice systems, and numerous private businesses and industries. Significant ethical and legal ramifications are associated with such testing. Those having positive test results may not receive employment, be dismissed from a job, be court-martialed, or suffer a damaged reputation.

To assure the integrity of workplace urine testing, two certification programs currently accredit forensic urine-testing laboratories. Laboratories conducting testing of federal employees are required to be certified under the Department of Health and Human Services Mandatory Guidelines for Workplace Drug Testing as published in the April 11, 1988, Federal Register (Department of Health and Human Services, 1988). The College of American Pathologists (CAP) also conducts a certification program for urine drug testing laboratories. The federal program regulates the process from specimen collection through testing to the reporting of results, whereas the CAP program allows flexibility in the construction of programs servicing a broad range of clients. Both programs involve proficiency testing and periodic on-site inspection of laboratories.

Forensic urine drug testing (FUDT) differs from other areas of forensic toxicology in which urine is the only specimen analyzed and testing is performed for a limited number of drugs and metabolites. Under the federal certification program, analyses are performed for a limited number of classes or drugs of abuse (Table 32-3). While FUDT laboratories may typically analyze 100 to 1000 urine specimens daily, only a relatively small number of those specimens are positive for drugs. To handle this large workload, initial testing is performed by immunoassays on rapid, high-throughput chemistry analyzers. A confirmation analysis in FUDT-certified laboratories is performed by GC-MS and LC-MS/MS.

Proper FUDT is a challenge to good laboratory management. As with all forensic activities, every aspect of the laboratory operation must be thoroughly documented—specimen collection, chain of custody, quality control procedures, method validation, testing, qualifications of personnel, and the reporting of results. The laboratory facility must be constructed and operated to assure total security of specimens and documents. Confidentiality of all testing results is paramount; only specifically authorized persons should receive the results. The presence of a controlled or illicit drug in a single random urine specimen is generally accepted as proof of recent or past substance abuse. However, positive urine drug findings are only evidence that, at some time before the collection of the sample, the individual was administered the drug, self-administered it, or was exposed to it. Positive urine tests do not prove impairment from drug abuse or addiction.

FUDT results are reported only as positive or negative for the drugs sought. Cutoff values are established for both the initial and confirmation assays (Table 32-3). The cutoff value is a concentration at or above which the assay is administratively considered positive. Below the cutoff value, the assay is reported to be negative for that drug or drug class. Obviously, drugs may be present below the cutoff concentration. However, the use of cutoff values allows uniformity in the drug testing and reporting of results. All test reports indicate the drug tested and its cutoff value. FUDT laboratories must be thoroughly familiar with all regulatory and analytical issues related to urine testing and devise strategies to resolve uncertainties.

Many individuals who are subject to regulated urine testing have devised techniques to mask their drug use either by physiological means such as the ingestion of diuretics or by attempting to adulterate the specimen directly with bleach, vinegar, or other products that interfere with the initial immunoassay tests (Warren, 1989; George and Braithwaite, 1996; Ferslew et al., 2003; Paul, 2004; Burrows et al., 2005). Thus, specimens are routinely tested for adulteration by checking urinary pH, creatinine, and specific gravity, nitrates, chromates, and noting any unusual color or smell. Recently, a mini-industry has developed to sell various products that are alleged to “beat the drug test.” These products contain chemicals that, when added to a urine specimen, interfere with either the initial or confirmatory drug test (Dasgupta et al., 2007; Jaffee et al., 2007; Larson et al., 2008). For example, several of these products contain glutaraldehyde, which will react with the nitrogen atoms of the antibody proteins of the immunoassay screening test, thereby cross-linking the antibodies and inactivating the assay. However, this disruption of the test is so complete that the immunoassay analyzer records almost no signal, thus indicating possible adulteration of the specimen. Another adulterant, for the marijuana metabolite urine test, contains sodium nitrite. In acidic urine, the nitrite salt is converted to nitrous acid which then converts the marijuana metabolites to nitroso derivatives, rendering them undetectable by routine GC-MS analysis (Lewis et al., 1999). Often, the pH of the urine is insufficiently acidic for complete nitroso conversion of the tetrahydrocannabinol (THC) metabolite, THC-carboxylic acid. Thus, the urine will screen positive by the initial immunoassay. When urine is acidified to extract the THC acid metabolite for MS confirmation testing, the metabolite is completely oxidized and undetectable. However, the deuterated THC–carboxylic acid added to the sample as the internal standard of the confirmation test is also completely destroyed. Failure to detect the internal standard readily alerts the analyst that an oxidant adulterant had been added to the urine. In such cases, a quantitative test for nitrite is performed. Nitrite may be present in urine from numerous internal and external sources such as foods, drugs, pathological conditions, and infection from nitrate-reducing microorganisms. However, none of these sources produces urinary nitrite concentrations that even begin to approach those obtained by the addition of adulterant amounts of potassium nitrite equal to 1000 mg/L (Urey et al., 1998). Most chemical adulterants can be detected in urine by specific colorimetric tests that can be readily adapted to high-volume auto-analyzers (Table 32-4). Thus, FUDT laboratories now routinely test not only for drugs of abuse but also for a wide variety of chemical adulterants. In most instances, a positive test result for adulteration has a consequence similar to a positive drug test.

There may be valid reasons other than substance abuse for positive drug findings, such as therapeutic use of controlled substances, inadvertent intake of drugs via food, and passive inhalation. For example, the seed of Papaver somniferum, poppy seed, is a common ingredient in many pastries and breads. Depending on their botanical source, poppy seeds may contain significant amounts of morphine. Several studies have demonstrated that the ingestion of certain poppy-seed foods results in the urinary excretion of readily detectable concentrations of morphine (ElSohly and Jones, 1989). Morphine is a major urinary metabolite of heroin. Therefore, to readily differentiate heroin abuse from poppy-seed ingestion, analysis may be performed for 6-acetylmorphine, a unique heroin metabolite (Fehn and Megges, 1985).

Even over-the-counter medications may present potential problems for laboratories conducting urine drug testing. Methamphetamine may occur as a racemic mixture of d- and l- optical isomers. d-Methamphetamine, a Schedule II controlled substance, is a potent central nervous system stimulant subject to illicit drug abuse, while l-methamphetamine (l-desoxyephedrine) is an alpha-adrenergic stimulant available in over-the-counter Vicks inhalers as a nasal decongestant. Cross-reactivity of l-desoxyephedrine with the initial immunoassay screening test may occur after excessive use of the Vicks inhaler (Poklis and Moore, 1995). Additionally, the most popular conformational GC-MS products for amphetamines are achiral. Therefore, if such analyses are performed, a “false-positive” result for d-methamphetamine may be reported. This dilemma is easily resolved if confirmation testing is done with a chiral GC-MS procedure, which can readily resolve the stereoisomers of methamphetamine (Fitzgerald et al., 1988).

Forensic toxicology activities also include the determination of the presence of ethanol and other drugs and chemicals in blood, breath, or other specimens and the evaluation of their role in modifying human performance and behavior. The most common application of human performance testing is to determine impairment while driving under the influence of ethanol (DUI) or drugs (DUID). Although operation of a motor vehicle is a common experience to most people, few appreciate the complexity of mental and physical functioning involved. A driver must simultaneously coordinate fine motor skills in tracking the road course and applying pressure to accelerator or brake—with visual attention immediately in front, to the horizon, and to the periphery of the vehicle—while continuously judging distance, speed, and appropriateness of response to signals, traffic, and unexpected events. The threshold blood alcohol concentration (BAC) for diminished driving performance of these complex functions in many individuals is as low as 0.04 g/dL, the equivalent of ingestion of two beers within an hour’s time to an average size person. The statutory definition of DUI in all 50 states is a BAC of 0.08 g/dL. These concentrations are consistent with diminished performance of complex driving skills in the vast majority of individuals. Over the past half century, an enormous amount of data has been developed correlating blood ethanol concentrations with intellectual and physiological impairment, particularly of the skills associated with the proper operation of motor vehicles. Numerous studies have demonstrated a direct relationship between an increased BAC in drivers and an increased risk of involvement in vehicular accidents (Council on Scientific Affairs, 1986). For example, alcohol impaired driving fatalities accounted for approximately one-third of all motor vehicle traffic fatalities in the United States in 2010 (National Highway Traffic Safety Administration, 2012).

During the past decade, there has been growing concern about the deleterious effects of drugs other than ethanol on driving performance. Several studies have demonstrated a relatively high occurrence of drugs in impaired or fatally injured drivers (White et al., 1981; Mason and McBay, 1984). These studies tend to report that the highest drug-use accident rates are associated with the use of such illicit or controlled drugs as cocaine, benzodiazepines, marijuana, and phencyclidine. However, most studies test for only a few drugs or drug classes, and the repeated reporting of the same drugs may be a function of limited testing. Before “driving under the influence of drugs” testing is as readily accepted by the courts as ethanol testing, many legal and scientific problems concerning drug concentrations and driving impairment must be resolved (Consensus Report, 1985). The ability of analytical methodology to routinely quantify minute concentrations of drug in blood must be established. Also, drug-induced driving impairment at specific blood concentrations in controlled tests and/or actual highway experience must be demonstrated.

The forensic toxicologist often is called upon to testify in legal proceedings. As a general rule of evidence, a witness may testify only to facts known to him or her. The witness may offer opinions solely on the basis of what he or she has observed (Moenssens et al., 1973). Such a witness is called a “lay witness.” In contrast, the toxicologist is referred to as an “expert witness.” A court recognizes a witness as an expert if that witness possesses knowledge or experience in a subject that is beyond the range of ordinary or common knowledge or observation. An expert witness may provide two types of testimony: objective testimony and “opinion.” Objective testimony by a toxicologist usually involves a description of his or her analytical methods and findings. When a toxicologist testifies as to the interpretation of his or her analytical results or those of others, that toxicologist is offering an “opinion.” Lay witnesses cannot offer such opinion testimony, as it exceeds their ordinary experience.

Before a court permits opinion testimony, the witness must be “qualified” as an expert in his or her particular field. In qualifying someone as an expert witness, the court considers the witness’s education, on-the-job training, work experience, teaching or academic appointments, and professional memberships and publications as well as the acceptance of the witness as an expert by other courts. Qualification of a witness takes place in front of the jury members, who consider the expert’s qualifications in determining how much weight to give his or her opinions during their deliberations.

Whether a toxicologist appears in criminal or civil court, workers’ compensation, or parole hearings, the procedure for testifying is the same: direct examination, cross-examination, and redirect examination. The attorney who has summoned the witness to testify conducts direct examination. Testimony is presented in a question-and-answer format. The witness is asked a series of questions that allow him or her to present all facts or opinions relevant to the successful presentation of the attorney’s case. During direct examination, an expert witness has the opportunity to explain to the jury the scientific bases of his or her opinions. Regardless of which side has called the toxicologist to court, the toxicologist should testify with scientific objectivity. Bias toward his or her client and prejudgments should be avoided. An expert witness is called to provide informed assistance to the jury. The jury, not the expert witness, determines the guilt or innocence of the defendant.

After direct testimony, the opposing attorney questions the expert. During this cross-examination, the witness is challenged as to his or her findings and/or opinions. The toxicologist will be asked to defend his or her analytical methods, results, and opinions. The opposing attorney may imply that the expert’s testimony is biased because of financial compensation, association with an agency involved in the litigation, or personal feelings regarding the case. The best way to prepare for such challenges before testimony is to anticipate the questions the opposing attorney may ask.

After cross-examination, the attorney who called the witness may ask additional questions to clarify any issues raised during cross-examination. This allows the expert to explain apparent discrepancies in his or her testimony raised by the opposing attorney. Often, an expert witness is asked to answer a special type of question, the “hypothetical question.” A hypothetical question contains only facts that have been presented in evidence. The expert is then asked for his or her conclusion or opinion based solely on this hypothetical situation. This type of question serves as a means by which appropriate facts leading to the expert’s opinion are identified. Often, these questions are extremely long and convoluted. The witness should be sure he or she understands all the facts and implications in the question. Like all questions, this type should be answered as objectively as possible.

Analytical toxicology in a clinical setting plays a role very similar to its role in forensic toxicology. As an aid in the diagnosis and treatment of toxic incidents, as well as in monitoring the effectiveness of treatment regimens, it is useful to clearly identify the nature of the toxic exposure and measure the amount of the toxic substance that has been absorbed. Frequently, this information, together with the clinical state of the patient, permits a clinician to relate the signs and symptoms observed to the anticipated effects of the toxic agent. This may permit a clinical judgment as to whether the treatment must be vigorous and aggressive or whether simple observation and symptomatic treatment of the patient are sufficient.

A cardinal rule in the treatment of poisoning cases is to remove any unabsorbed material, limit the absorption of additional poison, and hasten its elimination. The clinical toxicology laboratory serves an additional purpose in this phase of the treatment by monitoring the amount of the chemical remaining in circulation or measuring what is excreted. In addition, the laboratory can provide the data needed to permit estimations of the total dosage or the effectiveness of treatment by changes in known pharmacokinetic parameters of the drug or other chemical ingested.

Although the instrumentation and the methodology used in a clinical toxicology laboratory are similar to those utilized by a forensic toxicologist, a major difference between these two applications is responsiveness. In emergency toxicology testing, results must be communicated to the clinician within hours to be meaningful for therapy. A forensic toxicologist may carefully choose the best method for a particular test and conduct replicate procedures to assure maximum accuracy. A clinical laboratory cannot afford this luxury and may sacrifice accuracy for a rapid turnaround time. Additionally, because it is impossible to predict when toxicological emergencies will occur, a clinical laboratory must provide rapid testing 24 hours a day, every day of the year.

Primary examples of the usefulness of emergency toxicology testing are the rapid quantitative determination of acetaminophen, salicylate, alcohols, and glycol serum concentrations in instances of suspected overdose. Acetaminophen serum values related to the time after ingestion (see Chap. 33) not only indicate an overdose, but provide a prognosis for possible delayed hepatotoxicity and the need to continue administration of N-acetylcysteine antidote. In addition, continuous monitoring of serum values permits an accurate pharmacokinetic calculation of the ingested dose (Melethil et al., 1981). Similarly, salicylate serum values related to the time after ingestion may indicate an overdose, providing a prognosis for possible delayed severe metabolic acidosis and the need for lifesaving dialysis treatment. Continuous monitoring of serum salicylate values permits an accurate assessment of the efficacy of dialysis.

Ethanol is the most common chemical encountered in emergency toxicology. Although relatively few fatal intoxications occur with ethanol alone, serum values are important in the assessment of behavioral, physiologic, and neurological function, particularly in trauma cases where the patient is unable to communicate and surgery with the administration of anesthetic or analgesic drugs is indicated. Intoxications from accidental or deliberate ingestion of other alcohols or glycols—such as methanol from windshield deicer or paint thinner, isopropanol from rubbing alcohol, and ethylene glycol from antifreeze—are often encountered in emergency departments. Following ingestion of methanol or ethylene glycol, patients often present with similar neurological symptoms and severe metabolic acidosis due to the formation of toxic aldehyde and acid metabolites. A rapid quantitative serum determination for these intoxicants will indicate the severity of intoxication and the possible need for dialysis therapy. Alcohol infusion, in order to saturate the enzyme alcohol dehydrogenase, blocks the conversion of methanol and ethylene glycol to their toxic metabolites. Continuous monitoring of serum values not only permits an assessment of the clearance of the intoxicant by dialysis, but also assures a proper infusion rate of alcohol for effective antidotal concentrations (Fig. 32-7). To provide effective service to the emergency department, laboratories should have available chromatographic methods for the rapid separation and detection of alcohols and glycols (Edinboro et al., 1993).

The utilization of the analytical capabilities of a clinical toxicology laboratory has increased enormously in recent years. Typically, the laboratory performs testing not only for the emergency department but also for a wide variety of other medical departments, as drugs and toxic agents may be a consideration in diagnosis. Urine is analyzed from substance abuse treatment facilities to monitor the administration of methadone or other therapeutic agents and/or to assure that patients do not continue to abuse drugs. Similarly, psychiatrists, neurologists, and physicians treating patients for chronic pain need to know whether patients are self-administering drugs before such patients undergo psychiatric or neurological examinations. Analysis for drugs of abuse in meconium and urine obtained from neonates is used to corroborate the diagnosis of withdrawal symptoms in newborns and document fetal exposure to controlled substances. Toxic metal determinations, such as blood lead concentration, are often performed to assess possible toxic metal exposure or severity of toxicity. Analysis of heavy metals in 24-hour urine specimens is often used to rule out toxic metal exposure as a cause of symmetrical peripheral neuropathy prior to the diagnosis of neurological disorders such as Guillian–Barré syndrome. The clinical toxicology laboratory may often perform unique diagnostic tests that require sophisticated analytical capabilities such as GC-MS and LC analysis for organic acids and amino acids to detect inborn errors of metabolism in infants and children. Methods for the analysis of abnormal organic acids will also detect acidic drugs and other intoxicants such as salicylates, ethylene glycol, GHB, and valproic acid. Another such diagnostic test requiring sensitive chromatography is the timed metabolism of lidocaine to its monoethylglycinexylidide (MEGX) metabolite (O’Neal and Poklis, 1996). The rate of this conversion is a sensitive indicator of hepatic dysfunction and is often used to assess hepatic viability in donor livers prior to transplantation. MEGX formation may also be useful in monitoring the severity of the histological condition of patients with chronic hepatitis and cirrhosis (Shiffman et al., 1994).

Historically, the administration of drugs for long-term therapy was based largely on experience. A dosage amount was selected and administered at appropriate intervals based on what the clinician had learned was generally tolerated by most patients. If the drug seemed ineffective, the dose was increased; if toxicity developed, the dose was decreased or the frequency of dosing was altered. At times, a different dosage form might be substituted. Establishing an effective dosage regimen was particularly difficult in children and the elderly.

The factors responsible for individual variability in responses to drug therapy include the rate and extent of drug absorption, distribution, and binding in body tissues and fluids, rate of metabolism and excretion, pathological conditions, and interaction with other drugs (Blaschke et al., 1985). Monitoring of the plasma or serum concentration at regular intervals will detect deviations from the average serum concentration, which, in turn, may suggest that one or more of these variables need to be identified and corrected.

With multiple administrations of a given drug at regular intervals, plasma drug concentrations will gradually increase and eventually reach a plateau over the course of therapy. The plateau is referred to as a steady state condition, whereas the amount of drug absorbed is in equilibrium to the amount of drug eliminated. Dosage regimes are calculated so that plasma drug concentrations during steady state conditions are within the therapeutic range, and monitoring such conditions assures that an effective concentration is present. For drugs that have a defined correlation between serum values and undesired toxic effects, the lowest serum value immediately prior to dosing (trough) and the highest expected serum concentration (peak) are monitored to assure efficacy and minimize toxicity.

Because the drug being administered is known, qualitative characterization of the analyte generally is not required. Quantitative accuracy is required, however. Frequently, the methodology applied is important, particularly in regard to its selectivity. For example, methods that measure the parent drug and its metabolites are not ideal unless the individual analytes can be quantified separately. Depending on the drug, metabolites may or may not be active to a different degree than the parent drug. The cardiac antiarrhythmic drug procainamide is acetylated during metabolism to form N-acetylprocainamide (NAPA). This metabolite has antiarrythmic activity of almost equal potency to that of the parent drug procainamide. There is bimodal genetic variation in the activity of the N-acetyltransferase for procainamide, so that, in “fast acetylators,” the concentration of NAPA in the serum may exceed that of the parent drug. For optimal patient management, information should be available about the concentrations of both procainamide and NAPA in serum (Bigger and Hoffman, 1985).

Because absolute characterization of the analyte is not necessary for many drugs, immunoassay procedures are commonly used. This is particularly true of drugs with extremely low serum concentrations, such as cardiac glycosides, and drugs that are difficult to extract because of a high degree of polarity, such as the aminoglycoside antibiotics. In these cases, serum can be conveniently assayed directly by using commercially available kits for immunoassays.

The chromatographic methods in which an appropriate internal standard is added are favored when more than one analyte is to be quantified or if metabolites with structures similar to those of the parent drugs must be distinguished. Because the nature of drugs is varied, many different analytical techniques may be applied, including atomic absorption spectrophotometry for measuring lithium used to treat manic disorders. Virtually all the tools of the analyst may be used for specific applications of analytical toxicology. Drugs that are commonly monitored during therapy are presented in Table 32-5.

The analytical techniques employed by forensic toxicologists have continued to expand in complexity and improve in reliability and sensitivity. Many new analytical tools have been applied to toxicological problems in almost all areas of the field, and the technology continues to open new areas of research. Forensic toxicologists continue to be concerned about conducting unequivocal identification of toxic substances in such a manner that the results can withstand a legal challenge. The issues of substance abuse, designer drugs, increased potency of therapeutic agents, and widespread concern about pollution, and the safety and health of workers present challenges to the analyst’s knowledge, skills, and abilities. As these challenges are met, analytical toxicologists will continue to play a substantial role in the expansion of the discipline of toxicology.