Forensic Toxicology In Death Investigation

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If you have issues viewing or accessing this file contact us at 5Forensic Toxicology in Death InvestigationEugene C. Dinovo, Ph.D., and Robert H. CraveyForensic toxicology is a highly specializedarea of forensic science which requires expertise in analytical chemistry, pharmacology,biochemistry, and forensic investigation. Thepracticing forensic toxicologist is concernednot only with the isolation and identificationof drugs and other pOlsons from tissues, butalso with the interpretation of his findings forthe medical examiner, coroner, or other legalauthority.In our modern drug-oriented society theneed for the services of a toxicologist is clear.The benefits received from medication are sowell publicized that society tends to minimizethe dangers and pitfalls. The American peoplespend over 9 billion a year on drugs. In1971, the public spent approximately 5% billion on prescription drugs and about 3'12 billion for over-the-counter medications (Arena1974). It has been estimated that there are asmany deaths from drugs as from automobileaccidents. During a I-year period at theMontreal General Hospital, for example, 25percent of the deaths on the public medicalservice were the result of adverse drug reactions (Martin 1971). Estimates of deaths fromadverse drug reactions in the United Statesrange from 3,000 to 140,000 (Talley andLaventurier 1974).The cause of death in drug cases may rangefrom a clear and obvious overdose, often substantiated by a suicide note, to a minor drugrelated pathological process which, over anextended period, leads to a general decline inhealth. The latter situation is rarely recordedin mortality statistics.States, warrant an official investigation by thecoroner or medical examiner to determine thecause of death. The resolution of many legalquestions depends on the official pronouncement of the cause of death. The settlement ofinsurance claims often rests on the pronouncement of the death investigator. Accuracy in determining the cause of death dependson the cooperation and free flow of information among all members of the medicolegalinvestigative team: the police homicideinvestigator, the medical examiner's investigator, the forensic pathologist, the forensictoxicologist, and the medical examiner.The homicide investigator is usually thefirst to view the scene and, if he is properlytrained, it is he who maintains the sceneundisturbed for the medical examiner whomhe calls.The medical examiner's investigator is frequently the only member of the medicalexaminer's staff to actually view the sceneand talk to witnesses. He carries the mainbrunt of the investigation. He must obtainall information possible from the first officeron the scene, arrange for photographs of thebody and the scene to be taken, collect andpreserve all evidence including medicationsand empty containers found at the scene,interview all witnesses as well as family andfriends, and obtain a medical history fromfamily and/or attending physician. Severalexcellent references are available, in additionto chapters 2, 6, and 9 in the present book, toaid the investigator and the medical examiner:Medicolegal Investigation of Death (Spitz andFisher 1973), Homicide Investigation (Snyder1967), Techniques of Crime Scene Investigation (Svensson and Wendel 1972), and ThePathology of Homicide (Adelson 1974).The forensic pathologist performs the grossautopsy, collects the proper specimens foranalysis, and submits these specimens to theTHE MULTIDISCIPLINARY APPROA.CHTO DRUG DEATH INVESTIGATIONAbout 20 percent of all deaths occur incircumstances that, under the laws of most31

32DINOVO AND eRA VEYtoxicologist. Although gross findings in druginduced and drug-related deaths are oftennonspecific, e.g., visceral congestion andedema, discrete evidence suggesting poisoningby drugs has been documented (Svensson andWendel 1972; Adelson 1974; Siegel, Helpern,and Ehrenreich 1966; Helpern and Rho 1966;Helpero 1972; Siegel 1972; Garriott andSturner 1973; Citron et al. 1970; Hirsch1972).The forensic toxicologist is a crucial member of the tean1, and the objective laboratoryevidence he gathers must be considered, evaluated, and explained in the final assessmentof the cause of death.COLLECTION AND PRESERVATIONOF SPECIMENS FOR ANAL VSISThe evidence and information obtained bythe toxicologist is only as good as the qualityof his specimens. The proper specimens mustnot only be obtained uncontaminated, butmust also be preserved in their original condition for the toxicological analyses to bemeaningful. The human body is a dynamicorganism even in death, and metabolism,oxidation, and bactelial growth may contaminate, modify, or destroy substances ofinterest so that they cannot be detectedunless the specimens are properly preserved.The pathologist should confer with thetoxicologist concerning the choice and preservation of specimens, especially in cases requiring special treatment or exotic chemicalanalyses. Tissues other than blood shouldbe promptly frozen upon collection. As forthe blood sample, the toxicologist may preferthat it be collected in a chemically clean ora sterile container and maintained underrefrigeration to avoid hemolysis. Chemicalpreservation may interfere with some toxicological assays.It is recommended that samples of alltissues and fluids be ol)tained, placed inseparate containers, and properly labeled atthe time of autopsy regardless of the circumstances of the particular case. This procedurewill help the toxicologist in his search forpossible poisons throughout the body. It willalso prevent disinterment of the cadaver, withconcurrent toxicological problems caused bythe embalming fluid and decomposition if,due to new findings or history obtainedfollowing autopsy, a seemingly clear andstraightforward case suddenly becomes suspect.The specimen containers should be sealedwith a coroner's or medical examiner's sealand appropriate arrangements made fordelivery in order to maintain a valid chain ofcustody. A portion of each tissue must besaved by the toxicologist so that results of theanalyses can be corroborated by anotherlaboratory, should the occasion alise.The size of the tissue sample required forthe toxicologist to do his work will often bedependent on the instrumental capability ofhis laboratory. For example, if gas chromatography/mass spectrometry (GC/MS) with acomputer data system is available, smallquantities of each tissue may suffice. Conversely, if the laboratory is operating on asmall budget with little instrumentation, verylarge samples may be desirable.Fluids and Tissues Most Often AnalyzedThe tissues to be collected may be dependent upon the drug or other toxic substancesuspected. In any case involving the accidentalor intentional overdose of drugs, blood, gastlic contents, liver, bile, and urine (if available)should be considered minimal requirementsfor allalysis. Regardless of how well the onscene investigation is conducted, and ihethoroughness of the autopsy, precisely whattoxic compounds caused or contributed todeath is sheer speculation until the chemicalanalyses are complete. Therefore, a largequantity of each tissue or fluid is always preferable. If a storage problem exists, temporaryarrangements can usually be worked out withcommercial cold-storage firms to meet security requirements for a minimal cost.The choice of specimens and the quantityrequired do not pose apr ulem for the majormedical examiners' offices in the UnitedStates since these operations are contained ina central facility and the pathologist andtoxicologist are able to confer on each case.In a significant number of coroners' offices,autopsies are conducted in various hospitalmorgues and mortuaries and the tissues transported to laboratolies some distance away. It

FORENSIC TOXICOLOGY IN DEATH INVESTIGATIONis often difficult if not impossible for thepathologist and toxicologist to confer on eachcase. Table 1 is offer2d as a guide for thosepathologists to insure that adequate specimens are collected regardless of the nature ofthe case and the instrumental capability ofthe laboratory. As Adelson has pointed out(1974), when one is not sure what tissue tosave, the only safe approach is to save everything.Urine. Urine is a valuable fluid for thetoxicologist since it enables him to performsimple screening procedures such as spot testsand immunochemical tests for drugs or drugclasses, thus quickly informing him of theirpresence or absence in a certain concentration. Moreover, urine as the final depositoryof kidney drug excretion in many cases concentrates the dmg and metabolites to levelsthat are readily detectable. Drugs and metabolites may still be present in urine when theyare no longer detectable in the blood.Blood. Blood is valuable as the circulating,bathing medium of the organs when uncontaminated by other body or tissue fluids.Purity and cleanliness of the blood specimenare essential for the correct interpretation oftoxicological data. Contamination of theTABLE 1. Suggested tissue collection incases involving drugs(See also table 1 in chapter 3)Specimen 1QuantityBlood200 mlLiver500 gmBrain200 gmKidneyequivalent of oneBileall availableLung500 gmAdipose tissue50 gmGastric contentsall availableUrineall available1 In certain cases, other specimens such as vitreoushumor, hair, nails, etc., may be indicated.33specimen will render an already difficult taskimpossible or, worse, wUl lead to erroneousconclusions and interpretation. Two bloodsamples obtained from different body areascan serve as a check on each other and canprovide evidence for uniform distributionof the drug in the blood. The forensic pathologist should be discouraged from usingscooped-up or sponged-up "blood" from thebody cavity after autopsy. The left side ofthe heart may be a better source of bloodthan the right because of possible diffusionof the drug from the liver to the right side.Peripheral blood is perhaps the best singlesample.Liver. The liver is the maj or site of biotransfoIDlation in the body and, as such, itconcentrates many poisons and drugs. Poisonmay be detectable in the liver when none isdetectable in the blood. The major part ofthe liver should be saved for toxicologicalanalyses.Although the human is dead, the liver'smicrosomal metabolizing enzyme system willbe functioning and may well metabolize thedrug or agent of interest before measurementis possible unless the chemical reactions arestopped or slowed. The process may bestopped or slowed by freezing the tissueimmediately after autopsy and maintaining itin a frozen state until the assays can be performed.Stomach aud stomach contents. Often inoverdose cases the intact tablet.s or capsules ofdrugs are found in the stomach at the time ofautopsy and present a concentrated supply ofthe agent that can be readily identified. Evenwhen no tablets or capsules are seen, theirsolubilized remains on the stomach walls maystill present the best sample for identification.The total stomach contents, as well as thestomach, should be saved for analysis, and thetoxicologist should report the total quantityof drug recovered.Brain. Though the physiological action ofmany drugs lies in the brain, their concentration at this locus may not be very large.Nevertheless, many volatile poisons are retained by the lipid tissues of the brain andcan most readily be assayed there. Braincholinesterase should be assayed when organicpesticides are suspected (Curry 1969).Vitreous humor. The vitreous humor may

DINOVO AND eRA VEYprove useful for various clinical chemistryd( terminations (Siegel 1972; Garriott andSturner 1973; Citron et al. 1970; Hirsch1972; Curry 1969; Cae 1969; Coe and Sherman 1970; Sturnel' and Coumbis 1966; Coe1974) and may well be the specimen ofchoice for alcohol in certain instances (Stul'ner and Coumbis 1966; Coe 1974). Coe andSherman (1970) have found that chemicalchanges for many substances occur moreslowly in vitreous humor than in blood. Forcertain determinations, hemolyzed blood isunacceptable. Garriott (1974) has been ableto determine digoxin values more accuratelyusing vitreous humor rather than blood collected postmortem in coroner's cases.Kidney. Johnston, Goldbaum, and Whelton(1969) have found that morphine concentrations of 0.2 rng/100 gm or more were presentin the kidneys in case5 of sudden death causedby the intravenous use of heroin. They suggestthat drug levels in kidney tissue may be agood indieatol' of death that occurred rapidlyfollowing heroin injection. The kidney is alsoconsidered a tissue of choice in cases involvingheavy metals and sulfonamides.LUllg. The lung is a tissue of choice in casesinvolving inhalation of a drug. High concentrations of many drugs taken intravenously(for example, morphine) or orally (for exampIP, propoxyphene) may also be present.Bile. A number of important drugs, forexample, glutethimide and morphine, areeliminated through biliary excretion. In casesof prolonged survival time following heroininjPction, the bile may be the only specimenother than urine which can provide the analyst with a sufficient concentration of morphine for detection.Adipose tissue. Certain chemical compounds will accumulate in the fat and, inthose cases in which the victim has survivedfor some days following ingestion, this tissuemay offer the only proof of the compoundingested. Glutethimide (Goldbaum, Williams,and Johnston 1962), ethchlorvynol (Craveyand Baselt 1968) and thiopental (Goodmanand Gilman 1971) are among the drugs whichart' accumulated in adipose tissue. If a sampleof fat has not been collected by the pathologist, the peripheral fat from the kidney canbe analyzed.METHODOLOGICAL APPROACH TOIDENTIFICATION OF DRUGSThe onsite investigation and the autopsyfindings often provide the analyst with cluesto the possible offending agent. At the onsiteinvestigation, any evidence of drugs, pesticides, or other harmful agents should be collected and preserved. A thorough questioningof the victim's .social contacts can many timesprovide useful leads for the toxicological analysis. The astute investigator may save thetoxicologist many hours or days of effort.Reports of the onsite investigation and theautopsy findings should, therefore, be madeavailable to the toxicologist so that he mayuse pertinent information to minimize hisanalyses. When no evidence is found at thescene, and the autopsy shows no clear findings, a number of toxic substances must besearched for routinely, and the toxicologistis then presented with a general unknown. Itis the belief of many toxicologists that, if anadequate history were obtained and a complete onsite investigation and a thoroughautopsy were performed and followed bymicroscopic studies, the occurrence of generalunknown cases would be greatly minimized.The routine poison screen devised for generaluse will change from locality to locality depending, for instance, on the local drug subculture and whether an agricultural or urbancommunity is served.Separation of Drugs and Their MetabolitesFrom TissueAlthough some tests may be performeddirectly on specimens such as urine or gastriclavage, the majority are performed on organicsolvent extracts of body fluids or homogenizedtissues. Many methods exist for the isolationof drugs and their metabolites from blood andother tissues. Niyogi (1970) has published acomprehensive critical review of many ofthese methods. Ultimately, the selection of anappropriate means of extraction for screeningpurposes will depend on exactly which drugs,or groups of drugs, the toxicologist wishes toisolate. Most forensic toxicologists will extractthe specimen into organic solvents at differentpH's, thus separating into strong acids, w ak-,!J

FORENSIC TOXICOLOGY IN DEATH INVESTIGATIONacids, bases, and amphoteric drug fractions.Excellent references to this systematicapproach are found in Stewart and Stolman(1960, 1961), Sunshine (1969, 1971), Stolman (1963, 1965, 1967,1969), Kaye (1970),Curry (1969, 1972) and Clarke (1969).Other means of separating drugs and theirmetabolites from tissues or fluids includedistillation, digestion, and chromatographicmethods. In recent years, amberlite XAD-2polymeric adsorbent resin extractions havebeen widely used. This involves a one-stepapplication at pH 8.5 to isolate acidic, neutral,basic, and amphoteric drugs, though at lessefficiency than the usual organic solventextraction. Recovery can be improved forparticular classes of drugs by altering the pHat which the fluid is applied to the XAD-2column. A pH of 8.5 is often recommendedbecause it is optimal for morphine, thuscapable of identifying cases from methadonemab1tenance programs. Urine is applied to thewet column after being adjusted to pH 8.5and allowed to filter through the resin. Thedrugs are then eluted from their binding siteson the resin with ethylene dichloride, which isthen treated as the organic layer of a classicalextraction procedure.METHODS OF ANALYSISChromatographic techniques are mostoften used in the forensic laboratory for bothqualitative and quantitative tests for drugsand metabolites. Among these techniques arecolumn, paper, high pressure liquid, thin-layerand gas-liquid chromatography. Descriptionsof the latter two follow:Thill-layer chromatography (TLC) providesa simple, reasonably inexpensive, and sensitivemethod of analysis. Drugs are separated onthe basis of theIr molecular structure andproperties and may be identified using parameters such as Rfl value and reaction to a seriesof chromogenic reagents. Positive resultsshould not be based on one solvent systemalone; several systems, each yielding different1Rf distance traveled by substance from starting pointdistance traveled by solvent from starting point35Rf values for the drugs of interest, should beused. TLC methods are empirical, qualitative,and somewhat nonspecific. Many man-hoursof practice are necessary to acquire confidenceand expertise. In general, TLC is useful as ascreening tool. It is advisable to use otherindependent analytical methods in the forensiclaboratory before definitive identification isconcluded. Forensic scientists appear to bein agreement that a minimum of two differentparameters must be utilized for positive identification.Gas-liquid chromatography (GLC). Amongthe analytical tools available to the toxicologist, no single tool, probably, proves morE'useful than the gas chromatograph. It can provide a rapid, versatile, sensitive, and specificmeans for separating, identifying, and quantitating components of a complex mixture. Itcan offer a unique method for isolating acompound in question in pure form for identification by other means. Gas chromatographic columns of many different polaritiesand properties are readily available fro;.ncommercial sources or can be made in thelaboratory to accomplish almost any separation. A refinement of the GLC technique isthe formation of derivatives of the drug beforeinjection into the gas chromatograph. Derivative formation, an important identificationtechnique in classical organic chemistry, incombination with gas chromatography is apowerful tool in the toxicologist's repertoire.Moreover, some drugs may not optimallyseparate on GLC unless derivatives are made.Absol1Jtioll Spectrophotometry. Absorption spectrophotometry is a most usefulroutine tool in a toxicology laboratory. Avast amount of spectral data (visible, ultraviolet, and infrared) has been collected overthe past 25 years and provides a rich databank to be used by the toxicologist. Infraredspectrophotometry provides more information than either visible or ultraviolet, inasmuchas every chemical compound produces its owncharacte11stic spectrum, not unlike a fingerpdnt. However, purification of the unknowncompound prior to its introduction into theinfrared spectrophotometer is essential.Ultraviolet and visible spectrophotometryhave a greater practical application in theforensic laboratory than has infrared, in that

36DINOVO AND eRA VEYvaluable information can be obtained ofte

Forensic Toxicology in Death Investigation Eugene C. Dinovo, Ph.D., and Robert H. Cravey Forensic toxicology is a highly specialized area of forensic science which requires exper tise in analytical chemistry, pharmacology, biochemistry, and forensic investigation. The practicing forensic toxicologist is concerned

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