Barry Levine Office of the Chief Medical Examiner Stale of Maryland 111 PennSt. Baltimore, MD 21201 and Forenslc Toxicology Laboratory Armed Forces Institute of Pathology Washington, DC 20806-6000 Forensic toxicology is defmed by the American Board of Forensic Toxicology as the application of toxicology for legal purposes. The classic domain of the forensic toxicologist is the medical examiner's or coroner's office where analyses are performed on specimens from deceased individuals. The postmortem toxicology laboratory identifies and quantitates drugs or other foreign substances in biological specimens. Postmortem forensic toxicological analyses are well suited for suspected drug overdose cases because generally no identifiable lesions are found a t autopsy; a recent injection site or a large numher of unabsorbed tablet fragments in the stomach may be the only remarkable findings. Toxicological inquiries are also important in other death investigations. For instance, patient noncompliance with drug therapy becomes an issue in deaths resulting from seizure disorders and asthma: it may also be a fador in deaths of individuals being treated for depression or mental illness. Toxicological results are used by the pathologist along with fmdings from the autopsy and police investigation to assign a cause and manner of death. Over the past 25 years the field of forensic toxicology has expanded into other areas. For example, all states have laws prohibiting the operation of motor vehicles by people whose judgment has been impaired by their consumption of alcoholic beverages. State and local government laborato272 A
-
ries administer or regulate the testing of blood, urine, or breath for ethanol from drivers suspected of being intoxicated. Many states have analogous statutes forbidding the use of other psychoactive drugs when driving, and these same labs have been required to establish testing procedures for the presence of such drugs in blood and urine specimens. The field of forensic testing for drugs of abuse in urine samples has exploded during the past 10 years. Urine specimens may be collected for pre-employment screening, accident investigations, or identifieation of illicit drug use. The National Institute on Drug Abuse (NIDA) has formulated guidelines for laboratories performing drug testing of urine samples for federal agencies. These guidelines mandate methods for identifying and reporting five drug classes: amphetamineslmethamphetamines, cannabinoids, cocaine, morphinelcodeine, and phencyclidine. Speclmens The proper selection of specimens for analysis is an underrated but important aspect of the toxicological analysis. For instance, it is difficult if not impossible to acquire quality specimens after an autopsy has been completed; therefore, the pathologist must ensure that all necessary specimens are obtained at autopsy. Obviously, a varied choice of specimens is available in death cases. Blood should always be obtained. Ideally, two blood specimens should be collected, one from the heart and the other from a peripheral site such as the femoral or jugular vein. In certain situations such as motor vehicle accidents in which significant chest trauma has occurred, heart blood can be contaminated and the alternate blood specimen can be used for interpretation of results. Blood is also the specimen of choice if impair-
ANALYTICAL CHEMISTRY, VOL. 65, NO. 5, MARCH
1, 1993
ment caused by drugs is suspected in drivers or equipment operators. Blood collected for toxicological analyses should be preserved with sodium fluoride to limit microorganism formation and to enhance the stability of certain drugs. Urine has several advantages over blood. Many drugs andlor metabolites are present in higher concentrations in the urine than in the blood. Some classes of drugs also remain in the urine for days or longer following their use. Urine can be collected noninvasively, and skilled personnel are not required t o perform this task. The major drawback to urine screening is that no statement about impairment can be made. The presence of drugs or metabolites in urine is indicative only of prior exposure to the drug. In postmortem cases, urine (if present) can be collected directly from the bladder. Blood and urine are the two most versatile specimens in forensic toxicology. However, other specimens can he useful if acquired under certain conditions. Breath is an easily obtained specimen for ethrurol analysis. Vitreous humor should be collected in postmortem cases because it is more resistant to putrefactive changes than are other specimens. In overdose drug ingestions, analysis of stomach contents can provide easy identification of the substance or substances taken if intact tablets are present. Reeent work indicates that hair retains some drugs of abuse. For example, cocaine and heroin can be detected in hair. Because scalp hair grows a t a fixed rate of about 1 em per month, one can perform segmental analysis of the hair to establish a time frame of drug usage. The use of hair analysis in testing for drugs of abuse is still controversial; one major issue is the mechanism by which drugs enter hair. A related issue is the concern about external contami0003-2700~93/0365-272A/$04.W/0 0 1993 American Chemical SociePj
nation of the hair that may occur when illicit drugs are smoked. Ethanol Ethanol is the most frequently encountered drug in forensic toxicology. Among the major pharmacologic effects of ethanol are central nervous system depression, vasodilatation, hypothermia, and diuresis. Central nervous system depression plays a role in accidents; its severity can usually be correlated to the blood ethanol concentration. Complex brain functions are influenced even by low ethanol concentrations. If the blood ethanol concentration exceeds 400 mgldL, death can occur from respiratory depression. Reference tables list the effects of various blood ethanol concentrations ( I ) . As stated earlier, one complicating factor in the interpretation of postmortem blood ethanol concentrations is that after death a variety of microorganisms are capable of producing ethanol as well as acetaldehyde and n-propanol from various sugars or fatty acids. Urine and vitreous humor are two specimens resistant to this process and should be analyzed whenever a postmortem blood specimen is positive for ethanol. Detection of a positive blood ethanol concentration in conjunction with a positive vitreous humor and urine ethanol concentration would suggest antemortem ethanol consumption. Conversely, a negative vitreous humor and urine ethanol concentration would be indicative of postmortem ethanol formation in the blood. Analysis of blood and urine specimens for ethanol can be achieved by using a variety of methods. Enzymatic methods use alcohol dehydrogenase, which converts ethanol to acetaldehyde and nicotine adenine dinucleotide (NAD) t o NADH. The increase in absorbance a t 340 nm produced by NADH is directly pro-
portional to the amount of ethanol in the specimen. GC is used to analyze specimens directly after dilution with an aqueous internal standard or after being heated to 60 ’C in a sealed vial and sampling the vapor. Analysis of the head space permits quantitation without interference from the biological matrix. Multiple volatile compounds can be separated, identifed, and quantified by GC. A sample chromatogram of C,, C,, and C, volatiles is shown in Figure 1. Numerous methods are used for the analysis of ethanol in breath samples. The oldest breath-testing devices use the metal-catalyzed reduction of dichromate in sulfuric acid to chromic ion. Ethanol is oxidized in the process to acetic acid. The green color of the formed chromic ions can be monitored spectrophotometrically to quantitate the ethanol. An alternative breath-testing technology uses a fuel cell to measure the current generated by the electrochemical oxidation of ethanol to acetic acid. There are also breath-
tant to be aware of several factors. The physical and chemical characteristics of the drugs determine the separation and detection methods used. The disposition of drugs in the body is also critical. For example, in heroin abusers it is exceedingly rare to deted heroin in biological specimens; morphine is the predominant compound found. Similarly, two breakdown products of cocaine, benzoylecgonine and ecgonine methyl ester, remain in the body for much longer periods than does cocaine. Abused drugs include the “NIDA 5” drugs listed previously, barbiturates, benzodiazepines, and methadone. Therapeutic drugs also subject to abuse include over-the-counter and prescription medications. These encompass but are not limited to antiarrhythmics, anticonvulsants, antidepressants, antihistamines, narcotic analgesics, non-narcotic analgesics, and neuroleptic drugs. The initial step in the analytical screening for drugs is analyte separation. Except for some drug classes that can be analyzed directly in urine
ANALYrICAL APPROACH t e s t i n g devices t h a t use g a s chromatographic techniques. Recently, IR spectroscopy has become the most common analytical method used for breath-testing devices. Greater selectivity for ethanol is provided by including multiple wavelengths or a secondary detection system. Drug testlng Besides ethanol, a large number of other drugs are assayed in forensic toxicology laboratories. When establishing assays for drugs, it is impor-
specimens, the analytes of interest usually require separation from the biological matrix. Protein precipitation is useful for this purpose and is a relatively simple separation technique. Color testa and HPLC may be amenable to this separation method. The most common separation method used in forensic toxicology is liquid-liquid extraction. By using ionization and solubilitv characteristics, separation of basic“, neutral, and acidic drugs can be effected. Solidphase extraction with bonded silica columns is commonly used.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 5, MARCH 1,1993
-
273 A
ANALYTICAL APPROACH Once separation, if necessary, has occurred, the next step is toxicant identification using either spectrophotometry, chromatography, or an immunoassay. Color tests can be done directly on the specimen or on a protein-free filtrate of the specimen. Their ease of use makes these methods advantageous, especially a s screening tests. Salicylate and acetaminophen are two drugs for which relatively specific color tests exist. UV spectrophotometry was used with greater frequency in the past, but it lacks the sensitivity needed to detect therapeutic concentrations of many drugs encountered currently. Spectrophotometric methods also lack the specificity required to distinguish between parent drugs and their metabolites. This can be critical if the metabolites have varying degrees of pharmacologic activity. W has been used to identify and
Figure 1. Chromatogram of C, C, and C, volatiles. The inlernd Standard is 2 - b W n e . 10 m$L; the 2-m column is 0.2% Carbowax 1500 on Carbopack C; and the oven tempralure is 120 ‘C (isnhenal).
274 A
quantitate many drugs. In general, phenylmethylsilicone packing material with temperature programming can be used to separate a large group of substances (Figure 2). Flame ionization or nitrogen-phosphorus detectors are most commonly used. Benzodiazepines, sedative-hypnotic drugs containing halogens, can be determined a t very low detection limits with an electron capture detector. For polar and thermally labile compounds, HPLC is the preferred chromatographic technique. W detection is most desirable, but fluorescence, electrochemical, and refractive index detectors are also available. Electrochemical and fluorescence detectors permit greater detection limits; however, they lack the general applicability of W detectors. Immunoassays are based on the competition between the drug or drug class in the specimen and a labeled drug for sites on the antibody to that drug or drug class. A separation step may be required before measurement. These assays have several advantages over other techniques. They can be performed on urine specimens directly or on blood or tissue specimens after pretreatment and have good sensitivity to a particular drug or drug class. Three common immunoassays are enzymemultiplied immunoassay, radioimmunoassay, and fluorescence polarization immunoassay; each has advantages such as cost, amenability to high-volume automation, and antibody specificity. In order for a substance to be positively reported it is mandatory that a t least two different analytical techniques be used. The use of a s m n d or confirmatory technique is a fundamental principle in forensic toxicology. Numerous combinations of techniques (e.g., immunoassay and chromatography or spectrometry and chromatography) fulfill this requirement. Two different immunoassays would not be acceptable because of antibody similarities between commercially available kits. More defmitive confirmatory techniques provide structural information about the substance itself. GC/MS is currently the benchmark confirmatory technique used in the field. A gas chromatographic retention time plus a full-scan electron impact mass spectnun can be compared with a standard to provide conclusive identification in most but not all circumstances. For drugs present in lower concentrations, selected ion monitoring of three major
ANALYTICAL CHEMISTRY, VOL. 65, NO. 5, MARCH 1,1993
ions may be sflicient. GClIR spectroscopy also can provide conclusive identification in most situations. The interpretation of drug-testing results can be straightforward or complex. The presence of drugs and/or metabolites in urine will indicate exposure, but usually no assessment of impairment or toxicity is possible. Quantitation of drugs in blood is better correlated with toxicity or fatality. Blood quantitations must be interpreted in light of available history. A postmortem blood sample from an individual who was treated in the hospital for several days, which showed the presence of a drug in therapeutic concentration, may indicate much higher concentration a t an earlier time. A series of drugs, each present in therapeutic concentration, may produce synergistic effects when present in combination. A combination of ethanol and other central nervous system depressants, for example, can have potentiating effeds and cause death. Analytical approach The analytical approach taken by a forensic toxicology laboratory depends on the missions of that laboratory. If the lab is designed to perform only blood ethanol analyses, then a small setup with a single assay will be sufficient. For a large volume of samples with a limited number of analytes, a forensic urine drug-testing laboratory would perform immunoassay screens with confirmation by GC/MS if it were testing according to federal guidelines. In a postmortem forensic toxicology laboratory, where ethanol and therapeutic and abused drugs are assayed, methods should be established for the identification, confirmation, and quantitation of each group. The most common approach is to develop a protocol that comprehensively screens for alcohols and drugs. Once tentative identification has been made, more specialized testing can be done for confirmation and quantitation. No single analytical method is appropriate for all drugs; a series of methods in combination can provide the necessary breadth of coverage. For example, an d h l i n e extraction followed by GC with nitrogen-phosphorus detection a n d temperature programming can identify drugs within the following classes: antiarrhythmics, antidepressants, antihistamines, benzodiazepines, narcotics, neuroleptics, and sympathomimetic amines. Not all drugs within a class can be
detected in appropriate concentrations, and a toxicologist must h o w which drugs can be identified and at what detection limits. A weak acid extraction followed by GC or HPLC can identify acid and neutral drugs such as barbiturates, anticonvulsants, and glutethimide. A variety of commercially available immunoassays can identify amphetamines, barbiturates, benzodiazepines, cannabinoids, cocaine, opiates, and phencyclidine. Color tests are still useful for salicylate, acetaminophen, ethchlorvynol, and chloral hydrate (as trichloroethanol). Unusual cases In the vast majority of cases the routine analytical approach followed by a forensic toxicology laboratory is sufficient. However, there are times when unusual cases are presented that require special responses. The following examples illustrate the need for such special approaches. Fentanyl death (2). A critical component in death investigations is the analysis of the scene. In drug deaths, empty medication containers may provide useful information in
the ensuing toxicological analysis. In this case a 35-year-old anesthesiology resident was found dead in his apartment. At the scene were several syringes, an intravenous line, and a bottle of Versed (midazolam), a benzodiazepine often used in the induction of anesthesia. Subsequent analysis of the blood showed the presence of midazolam, but at a subtherapeutic concentration. Fortunately, one of the syringes still contained some material for analysis. Analysis of the syringe by GC/MS revealed fentanyl, a potent nareotic also used in operating moms. Its presence in the blood at ng/mL concentrations would not he picked up by routine toxicological screening. Further analysis of the blood and tissues by GC/MS resulted in the detection of fentanyl; the medical examiner ruled that the cause of death was fentanyl intoxication. USS Iowa diwtex (3). The USS Iowa was engaged in a gunnery exercise on April 19,1989,when an explosion occurred in the number two turret, killing 47 seamen. The signihmt toxicological question was: Which deaths occurred from the explosion
I
!’
Time rigure 2. Chrornarograrn OT an aiMllne-exrraaaDle arug sranaara. A DE-5 column (15 m v 0.25 mm i.d.. 0.25ym film thickness) was used. The oven 1emperatYre began ai 110 ‘Cfor 1 min. increased by 20 ’C per min 10 200 ‘C.then increased by 10 ‘C per min io 280 ‘C, holding for 15 min. (1) amphetamine, (2) methamphetamine, (3) nicotine, (4) pseudoephedrine. (5) meperidine, (6) lidocaine, (7) phencyclidine, (8) chlorphentramine. (9) methadone. (10) amitriptyline. (11) nortriptyline. (12) codeine. (13) diazepam, (14) nordiazepam, (15) chlorcquine, (16) alpwolam, (17) fhioridazine.
and which deaths occurred from the resulting fire? In the case of fire, CO or burns usually cause death. CO binds tightly to hemoglobin, the oxygen transport vehicle for the body, limiting the delivery of oxygen to the tissues. This causes hypoxia and death. If death occurred from the explosion and before the fire, a normal blood carboxyhemoglobin would be measured. Conversely, an elevated carboxyhemoglobin would suggest survival in the initial explosion but death in the fxe. Ten of the 47 fatalities had carboxyhemoglobin saturation values less than or equal to lo%, which is considered normal. Eight of those deaths were attributed to blunt force injuries, and the two others were caused by a combination of blunt force and thermal injuries. On the other hand, of the 37 victims with elevated carboxyhemoglobin,30 were judged to have died as a result of the fire.
Attempted murder with pancuronium (4). Pancuronium bromide is a nondepolarizing neuromuscular blocking agent that acts by competing with acetylcholine for cholinergic receptors at the motor end plate. It is a quaternary amine, which makes its separation from biological matrices a difficult analytical challenge. In this case, a nurse was accused of attempting to murder her husband by twice administering to him a n unknown neuromuscular blocking agent. On both occasions the husband was hospitalized, and his urine was collected within 1-2 h of the alleged injections. These specimens were screened using Rose Bengal dye, which forms a fluorophore with pancuronium but not with other quaternary amines. For confirmation, an ion-pair extraction followed by thin-layer chromatography was performed. The spot corresponding to pancuronium was scraped, extracted, and separated using GC/MS. Although pancuronium does not show a peak, a thermal breakdown product that does is produced ifthe injection port temperature is sufficiently elevated. The drug was confirmed using selected ion monitoring. The accused pleaded nolo contendere to the charge of attempted murder. Phenobarbital in maggots (5). In badly decomposed bodies the acquisition of common biological specimens is often impossible. The decomposition process often leads t o infestation of the remains with mag. gots. In this case, the body of a
ANALYTICAL CHEMISTRY, VOL. 65, NO. 5, MARCH 1 , 1 9 9 3 * 275 A
CLASSIFIED HELP WANTED
RESEARCH CHEMIST/ MINERALS RESEARCH
an excellent academic record w h i i h should indude a MS in Chemistry Or OWChemistN.
indlude salary history to: Cdlte Corpomtbn, P.O. Box 519, Lornwc, CA 934384519, E 0 E., MIFID
HELP WANTED ADS ROP display at ROP rates. Rate based on number of insertions within contract year. Cannot be combined for frequency. unn i-n 6-n 1z.n 1" (25mm)
$210
$190
$le4
zcn
48.17
72.~1
$170
$160
$150
CALL OR WRITE JANE GATENBY
ANALYTICAL CHEMISTRY 1599 Pod Road East P.O. Box 231 Westport, CT 08881 203-258-8211 FAX: 203-256-8175 LABORATORY SERVICE CENTER (Equipment. Materials. Services. instruments lor LeBsing). Maximm spece 4 inches per adwltisement. Column width. 2-3/16': two wIumn width, 4-9/16'. Artwork accepted. No wmbimtion of directory rates wiih ROP advertising. Rates based on number of inches used wlhin 12 months from first date of first insertion. Per inch: 1 " $185; 12. $180; 24.$175; 36" -$170; 46" -$165.
-
-
-
ANALYTICAL CHEMISTRY 1599 P a t Road East ~. .-. .. ... FAX: 205-258-8175
FREE DATA, FAST To quickly amass data on a11 of the prod-
ucts you need, consult the Lab Data Service Section on our Analytical Chemistry reader rqply card insert.
ANALYTICAL APPROACH 22-year-old female was found badly decomposed and skeletonized in a wooded area. An empty container, which showed a recently filled prescription for phenobarbital, was found in a purse next to the body. There was a history of previous suicide attempts, and a suicide note was found at the scene. The only available specimens for analysis were the insect larvae. The larvae were homogenized and the proteins precipitated and extracted with chloroform. The extract was concentrated and iqjected into a gas chromatograph. Phenobarbital was identified by retention time and quantitated at 100 mglkg; confirmation was made by GCIMS. The entomologist involved in the case coucluded that the phenobarbital had originated from the tissues consumed by the maggots. Lidocaine homicides (6). In 1981 a series of deaths within a twomonth period occurred in a California hospital intensive care unit. Many of the patients experienced seizures, respiratory arrest, and bluish discolorations in the thoracic region. When a similar death occurred at another hospital, a review of personnel records indicated that one nurse was employed at both hospitals and had access to all of the deceased patients. Postmortem toxicological analysis of some of the decedents indicated massive amounts of lidocaine in the fluids and tissues. The toxic effects of lidocaine are consistent with the symptoms displayed by the dying patients. The n u r s e was charged with and subsequently convicted of 12 of the killings. Morphine intoxication (7). A 26year-old woman diagnosed with acute leukemia was admitted to the hospital. Her extremely high fever was treated with antibiotics. When her heart activity stopped, she was successfully resuscitated and placed on a respirator. Two days later, the respirator was stopped and 90 mg of meperidine allegedly was administered. Two hours later, the patient died. Subsequent investigation indicated t h a t a 90-mg dose of morphine rather than meperidine had been given. Morphine is 7-10 times more potent than meperidine, as a pain killer and as a respiratory depressant. Four months after the death, the body was exhumed for autopsy and collection of specimens for toxicology. The liver, muscle, gall bladder, and kidney were negative fol meperidine: however. hieh concen. traiions of morphine were Found in the
!76 A * ANALYTICAL CHEMISTRY, VOL. 65, NO. 5, MARCH 1, 1993
brain, liver, and muscle. The physician who administered the morphine was charged with manslaughter but was acquitted after a long trial. Summary This article has briefly highlighted some aspects of forensic toxicology, including specimen collection, the identification and measurement of analytes, and the analytical approach that should be followed to generate toxicological results. The role of a forensic toxicology laboratory may be varied. It can be asked to assign a cause of death or to explain behavior in deaths of known cause. Ita findings may also be used to restrict or deny driving privileges. In addition, positive results from a drug screening may provide a reason to refuse employment or to remove an individual from his or her job. Regardless of the intended use, the results must be generated in a n accurate scientific manner.
References (1)Sunshine, I. In Methodoloafor Analyt'. cal Toxicolo Sunshine, I., Ed.; CRC Press: ClevTtd, OH, 1975;p. 148. (2) Levine, B.; Goodin, J. C.; Caplan, Y. H. Forensic Sei. Int. ism,45,247-51. (3)Mayes, R.; Levine, B.; Smitb, M. L.; Wagner, G.N.; Froede, R. J. Forensic Sci. 1992,37,1352-57. (4)Brigha, E. J.; Davis, P. L.; Katz, M.; Dal Cortivo, L. A. J F0rew.c Sci. ISSO, 35, 1468-76. ( 5 ) Beyer, J. C.; Enos, W. F.; Stajic, M. J Form'c Sci. lSBO,25,411-12. ( 6 ) Starrs, J. Scientific Sleuthing Review 1992,16,1-5. (7) Worm, I&;Steentaft, A; Christensen, H. J Forensic SCI.Soc. 1982,23,209-12.
Bany Levine received a B.S. degree in chemistry from h p l a College in Baltimore, MD, in I978 and a Ph.D. in pathologyftvm the Medico[ College of Virginia in 1982. In I983 he began work as an as&ant toxicologist at the Ofice of the Chief Medical Examiner (OCME), State of Maryland. He became chiefof the Forensic Toxicology Laboratory fir the Armed Forces Medical Eraminer at the Armed Forces Institute of Pathology (AFIP) in Washington, DC, in 1989. Recently he returned to OCME as a toxicologist while retaining a pari-time position at AFIE He also is a clinical associate profmor of pathology at the University of Maryland at Baltimore.
-
