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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.
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Case History and Specimens
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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).
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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).
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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.
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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.
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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.
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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).
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Toxicological Analysis
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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.
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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.
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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.
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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.
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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.
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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).
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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.
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Interpretation of Analytical Results
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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.
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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).
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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.
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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.
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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.
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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.
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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).
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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.
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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.
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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).