Principles and Procedures in

Forensic Toxicology
John F. Wyman, PhD

KEYWORDS
• Review • Drugs • Analysis • Forensic • Toxicology • Procedures

KEY POINTS
• Forensic Toxicology is composed of Postmortem Toxicology, Human Performance Toxi-
cology and Drug Urinalysis.
• Forensic Toxicology results have the potential of being scrutinized in court; as a result,
testing is more comprehensive, with greater emphasis on specificity and accuracy in
identifying potential toxicants.
• Conclusions about postmortem results must be made after considering all aspects of a
case, including medical records, matrices analyzed, drug interactions, drug tolerance,
postmortem interval, and the like.

INTRODUCTION

Forensic toxicology concerns the application of toxicology to situations that may have
medicolegal review, and as a consequence, results must stand up to scrutiny in a
court of law.1 There are primarily three subdisciplines of forensic toxicology:
1. Postmortem toxicology, more recently referred to as death investigation toxicology.
2. Behavioral or human performance toxicology, which concerns
a. Impaired driving as a result of alcohol and/or drugs consumption.
b. Drug-facilitated sexual assault cases.
c. Doping control. Screening of athletes for performance-enhancing substances is
monitored by the World Anti-Doping Agency.2 In this category must be included
equine and canine toxicology testing, because entire laboratories are dedicated
to this specific purpose.
3. Forensic workplace drug testing or drug urinalysis, which is performed as a
preemployment and/or random monitoring of employees for illicit drugs or court-
ordered testing of convicted drug offenders.

The author has nothing to disclose.

Toxicology Department, Cuyahoga County Region Forensic Science Laboratory, Cuyahoga
County Medical Examiner’s Office, 11001 Cedar Avenue, Cleveland, OH 44106, USA
E-mail address: [email protected]

Clin Lab Med 32 (2012) 493–507

http://dx.doi.org/10.1016/j.cll.2012.06.005 labmed.theclinics.com
0272-2712/12/$ – see front matter Published by Elsevier Inc.
494 Wyman

Closely related but in a category of its own is forensic drug chemistry.3 This
discipline is concerned with drug and chemical analysis, as is toxicology, but in regard
to nonbiological specimens such as seized bales of marijuana, packets of synthetic
cannabinoids, pills, “meth lab” reagent analyses, rocks of crack cocaine, and the like.
Toxicology and drug chemistry laboratories often work together on different aspects
of a case, especially when the laboratories are housed in the same facility. An
important consideration when laboratories are in the same building is that extraction
of drugs by each discipline must be accomplished in different rooms to avoid possible
contamination of toxicology specimens by the relatively massive quantities of drug
chemistry specimens.
To assist with the practice of forensic toxicology, a guide as to how the discipline
should be performed is provided in the form of forensic toxicology laboratory
guidelines, prepared by the Society of Forensic Toxicology (SOFT) and the Toxicology
Section of the American Academy of Forensic Sciences (AAFS).4 Also, at the time of
this writing, a “draft” document entitled Scientific Working Group for Forensic
Toxicology (SWGTOX) Standard Practices for Method Validation in Forensic Toxicol-
ogy has been released for public comment. The practice of urine drug testing is
defined by Mandatory Guidelines for Federal Workplace Drug Testing Programs,
issued by the Department of Health and Human Services.5 This review is restricted to
the subdisciplines of postmortem and behavioral toxicology.

HOW FORENSIC TOXICOLOGY DIFFERS FROM CLINICAL TOXICOLOGY

Clinical toxicology is typically hospital-based, with the emergency room physicians

being the primary customers. Analytical results must be obtained with speed to help
confirm the therapeutic regimen for living patients, and an initial screen result by itself
is sufficient for use in medical evaluation. Clinical analysts rarely are called about
judicial matters, compared with forensic analysts. Quantification and confirmation of
drug findings are not usually relevant to the treatment and often are not possible
because of time constraints. Analytical instrumentation such as immunoassay pro-
vides fast but less specific results at a relatively lower cost. In larger laboratories with
high-end analytical capability, physician-ordered therapeutic drug monitoring is
performed.
By comparison, forensic toxicology can move at a slower and more comprehensive
pace. Critical to the analytical process is a rigorous chain of custody from which the
identity of the specimen is certain. Because the results for any case have the potential
to end up in a court of law, there is a greater emphasis on specificity and accuracy in
identifying potential toxicants. The cost of analysis is generally higher. For results to
be reported, both a screen and confirmation must be performed using two different
analytical procedures wherever possible; alternatively, when two analytical proce-
dures are not available, a confirmation may be accomplished using (a) two different
methods for extraction, (b) by demonstrating that a specific drug (metabolites) is
present in two different specimens (eg, blood and urine), and/or (c) the drug is listed
as part of the case history from the standpoint of medical record, prescription record,
or death investigation.

CERTIFICATION AND ACCREDITATION

Forensic toxicology and all fields of forensic science are currently experiencing a
period of accelerated change. The impetus for change came in 2009 with the National
Academy of Sciences report6 on the need for overhaul of forensic sciences in the
United States. A major recommendation of this report was that forensic scientists
 Forensic Toxicology 495

should be certified in their specific discipline and laboratories should be accredited.

Forensic toxicology certification is available primarily through two organizations: The
American Board of Forensic Toxicology (ABFT)7 and the Forensic Toxicology Certi-
fication Board.8 Other certifications include the American Board of Clinical Chemistry,
the American Board of Toxicology, the American Society for Clinical Pathology, and
the National Registry of Certified Chemists. In addition to certification of analysts,
certain states require that individuals performing toxicology testing on specimens
from impaired drivers be licensed by the state’s health departments.
Accreditation of toxicology laboratories can be accomplished through The ABFT
and/or, based on the ISO 17025 Standards (Testing and Calibration Laboratories),
through the American Society of Crime Laboratory Directors Laboratory Accreditation
Board (ASCLD/LAB) program9 or Forensic Quality Services, Inc.10 Currently New
York, Texas, and Oklahoma are the only states that require that toxicology laborato-
ries be accredited. Other accrediting bodies include the National Laboratory Certifi-
cation Program under the Substance Abuse and Mental Health Service Administra-
tion,11 the National Association of Medical Examiners (NAME),12 and the College of
American Pathologists (CAP).13

TOXICOLOGY TESTING
Chain of Custody
In that the results of forensic toxicology testing may be used in court proceedings, the
first necessary component of the testing process is to demonstrate the validity of test
specimens. This demonstration is accomplished through the chain of custody,14
which documents the chronologic disposition and condition of specimens from the
time of collection to the time of disposal. The person initiating the chain of custody
would typically provide the identity of the individual from whom the specimen was
collected, what the specimen is, when it was collected (time and date) and by whom,
including signatures. Tamper-evident tape with initials across the tape may be used
to help maintain the integrity of the specimen. As the specimen moves through the
transfer and testing process, printed names and signatures of releasing and receiving
persons are recorded as are the time and date, the condition of the specimen, and the
reason for transfer. Without correct, legible, and intact chain of custody documenta-
tion, the integrity and security of the specimens cannot be established, and the results
of toxicology testing may be judged to be inadmissible to the court. The chain of
custody form is often combined with the toxicology request form into a single
document. The toxicology request form allows the selection of specific testing
batteries such as a volatile screen, drug of abuse screen, comprehensive analysis, or
other special testing requests.

Testing Service Provided

A standard testing battery within a forensic toxicology laboratory will include an
alcohol (volatile) screen, a drug of abuse screen, electrolyte profile, and a compre-
hensive analysis. All screens include confirmation and quantitation of any positive
results. Volatile analysis is most commonly performed by gas chromatography (GC),
whereas drug of abuse screens are performed with immunoassay. Immunoassay is
continually expanding to allow screening for tricyclic antidepressants, salicylate,
acetaminophen, methadone, oxycodone, barbiturates, carisoprodol, promethazine,
and other drugs. Some laboratories are transitioning to use of high-performance liquid
chromatography (HPLC)/mass spectrometry (MS) for drug of abuse screens. Confir-
mations are most often performed by GC/MS analysis with or without derivatization
of analytes or with HPLC/MS/MS, if this instrument is available. A comprehensive
496 Wyman

drug screen will include a volatile assay, an immunoassay of blood and urine, followed
by GC/MS analysis of extracts of blood and urine. Electrolyte analysis is performed on
vitreous humor rather than blood. Electrolyte values in blood are difficult to interpret
after death, whereas sodium, chloride, magnesium, calcium, urea nitrogen, and
creatinine remain essentially unchanged in the early postmortem period in the
vitreous. Elevated glucose will persist in the vitreous allowing detection of hypergly-
cemia, whereas normal glucose levels fall and have no interpretive value. Other
analytical procedures provided by laboratories fall into the category of “case-
directed” testing. These are tests that are not routinely performed but are necessary
based on the circumstances of the case. Examples include (1) carboxyhemoglobin
assay in victims from house fires or decedents found in automobiles, (2) antipsychotic
batteries for schizophrenics and manic/depressives, (3) anticonvulsant testing for seizure
histories, (4) heavy metal assay for alleged poisonings, (5) gamma-hydroxybutyric acid in
drug-facilitated sexual assault cases, and ethylene glycol as indicated. To complete case
analysis, evaluation of evidence (patches, contaminated clothing, pill from gastric
contents, drug paraphernalia (spoons, syringes, glass mirrors, wire mesh, and so forth) for
drugs should be performed. If toxicology and drug chemistry are housed in the same
laboratory area, evidence items can be analyzed by drug chemistry.

Matrices
The typical matrix for workplace or court-ordered drug testing is urine, although use
of alternative matrices (sweat, hair, and/or saliva) are being examined to with
increasing frequency.15 Behavioral toxicology most frequently is performed on blood
and/or urine specimens. Blood for forensic analysis is collected in gray top tubes
containing the antimicrobial additive sodium fluoride and potassium oxalate as an
anticoagulant. In that results may be presented in court, collection of blood in gray top
tubes is an important forensic consideration. An often used defense strategy is to
question the integrity of the specimen from the standpoint of in vitro production of
ethanol. This argument is moot if the specimens are collected in gray top tubes and
refrigerated. Interpretation of impairment from drug levels in urine is not possible,
although some states have per se driving laws based on the concentration of drugs
in urine. Whether the individual was under the influence of drugs or not cannot be
established from urine results; all that can be reasonably known is that the individual
was exposed to the drug.

Postmortem
Postmortem specimens are many and quite varied. If only an external examination is
performed (no autopsy), then blood, urine, and vitreous humor are typically collected.
During an autopsy, blood, urine, vitreous, bile, gastric, liver, spleen, kidney, brain,
muscle, and hair may be collected. For fluids that have a finite volume such as gastric,
bile, and urine, it is important to record the total volume present so that the total
amount of drug can be calculated for the specific compartment. Other specimens that
may be collected depending on the condition of the body and the toxicant of interest
include nails, bone, meconium, cerebrospinal fluid, and blood clots. If the clot is
connected to the cause of death, such as traumatic head injury with subdural
hematoma, the clot should be homogenized and analyzed. This procedure will give
some indication of blood constituents immediately following the time of the injury.

Hospital Specimens
Considerations for blood clots are similar for hospital specimens. If the decedent
arrived at the hospital and admission specimens were collected, these are the most
 Forensic Toxicology 497

important specimens to analyze to obtain a valid interpretation of drug or alcohol

contributions to the cause of death. Conversely, if admission specimens are not
available and the hospital record reflects an extended hospital stay and/or transfu-
sion/hemodilution, performing toxicology on postmortem specimens may be judged
to be of little value. If family members indicate that they believe the hospital caused
their loved one’s death, then it is important that the laboratory analyze both admission
blood (if available) and postmortem blood. Most hospitals keep collected specimens
for a short time (eg, 1 week) before they are discarded. Blood banks may hold
specimens for a longer period than the hospital. Death investigators are trained to act
quickly to recover hospital specimens, if they are available. In some situations,
hospitals may work with coroner/medical examiner offices to preserve blood when a
medicolegal investigation is expected.

Other Analytical Issues for Consideration

1. First assay. The first test performed in a forensic analysis should be a screen for
volatiles, in that ethanol, acetone, methanol, and other volatile constituents will be
depleted in concentration each time the stopper is removed from the blood,
vitreous, or urine tube.
2. Dedicated instrumentation. If the laboratory can afford the luxury of instruments
dedicated to specific assays, this is an efficient way to operate. Time spent
changing columns and loading new methods is avoided so that the flow of
analytical assay is more productive.
3. Drug stability. Certain analytes (olanzapine, promethazine,16 chlorpromazine,
chlorprothixene, thioridazine, triflupromazine, ziprasidone,17 zopiclone,18 cya-
nide,19 synthetic cathinones,20 cocaine21) should be measured as soon as possible
because of instability in storage. It is a good practice to characterize the stability of
analytes as part of the validation of the analytical procedure. In most cases, stability
is improved by refrigeration or freezing and by use of gray top tubes containing
sodium fluoride.
4. New drugs. Laboratories must stay current with the constantly changing menu of
available drugs, both prescribed and illicit. This ongoing task can be readily
accomplished by attending annual meetings (SOFT, AAFS, The International
Association of Forensic Toxicologists, NAME) and subscribing to relevant
journals (Journal of Analytical Toxicology and the Journal of Forensic Sciences).
SOFT’s quarterly newsletter (Tox Talk) is an online publication containing a
“New Drug” section.
Additional requirements are
a. To obtain new drug standards. Commercial drug companies include Cerilliant,
Lipomed, Cayman Chemicals, and Grace Chemicals, to name a few. If a drug is
not available it can sometimes be obtained by contacting the pharmaceutical
company that manufactures the drug.
b. GC/MS libraries must be updated continually. Commercial libraries appropriate
for drug analysis including PMW (Pfleger, Maurer, Weber), NIST (National
Institute of Standards and Technology), and Wiley Registry are available but
expensive to acquire. Additional sources that are currently free are the RTI
International “Forensic DB” (adopted from the AAFS library made available by Dr
Graham Jones),22 Scientific Working Group Drug library,23 and of course,
creating in-house libraries from acquired standards.
5. In GC and liquid chromatography (LC) analyses, to detect the possibility of
carryover from one injection to the next, it is good practice to insert solvent blank
498 Wyman

injections between control and case extracts. With autoinjectors, this process is
easily accomplished using the instrument sequencing program.
PRINCIPLES OF QUALITY ASSURANCE

A quality assurance (QA) program is required to ensure that the laboratory produces
consistently reliable drug/chemical identification and quantitation. Aspects of a QA
program include
1. Competent analyst with access to continuing education. The number of analysts must
be sufficient to handle the workload and provide testing services requested. If
resources allow it, each analyst would ideally attend a national or international meeting
each year. Certification in forensic toxicology should be encouraged. Subscription to
relevant journals and acquisition of up-to-date toxicology texts are needed.
2. An adequate work environment with properly maintained instrumentation. Docu-
mentation of maintenance and corrective actions is required. A planned, systematic
replacement of obsolete instruments should be incorporated into the laboratory
budget.
3. Appropriate documentation of policies and procedures is required. This documen-
tation is usually maintained as standard operating procedure manuals for admin-
istrative policies, quality assurance, analytical methods, accessioning of speci-
mens (and disposal), training, safety, corrective actions, improvements, personnel
training and competency, and the like.
4. Proficiency testing for volatile analytes and for drugs in blood, serum, and urine
specimens is required. Typically, laboratories will subscribe to the CAP proficiency
tests,24 which will include series AL1 (American Association for Clinical Chemistry/CAP
whole blood alcohol/ethylene glycol/volatiles, or AL2 (same but in serum), and forensic
toxicology– criminalistics (FTC) and toxicology (T). These series require that the
laboratory conduct proficiency testing three times each year for each series. For
accreditation by the ABFT the AL1, FTC, and T series test must be performed.
5. Validation of new analytical methods prior to their use in case work may include the
following criteria:
a. Limit of detection (LOD), which is the lowest concentration detectable above
background noise; usually three masses are used for confirmation.
b. Limit of quantitation, which is normally the lowest concentration determined for
the calibration curve.
c. Linear range for an analyte is needed to establish the upper and lower
concentrations at which the instrument response directly corresponds to the
concentration of the drug/chemical. Determinations that exceed the linear range
of the calibration curve must be repeated after the specimen has been appro-
priately diluted. Values that are less than the lowest concentration of the
calibration curve, and greater than the LOD, are normally reported as less than
that concentration or as “trace.” Nonlinear relationships (eg quadratic) may exist
for specific analytes, and use of instrument software to find the “best fit” curve
will improve analytical accuracy.
d. Accuracy is assessed through the use of controls. In order to validate the
calibration curve, control concentrations must be analyzed. Preparation of
controls should be from a source different from the source used to prepare the
calibration curve, or else the curve will most assuredly be validated whether it is
accurate or not. As much as is possible, calibrators and controls should be
“matrix matched” (prepared in drug-free blood or urine). Depending on the
assay, an acceptable accuracy may be as much as ⫾ 20% from the target
value. Volatile analysis of ethanol should not vary more than ⫾ 5% of the
 Forensic Toxicology 499

target value. Along with positive controls it is imperative that matrix-matched

negative or blank controls be run with each assay. Calibration curves should be
prepared with at least three different concentrations that define the linear range
(low, middle, and high). Controls should be prepared at both high and low
concentrations relative to the calibration curve, and both high and low controls
should be included at the beginning and at the end of a batch analysis. A good
practice is to include calibrations curve and control samples with each batch
analysis, although some laboratories may make use of “historical” curves and
freshly prepared controls.
e. Precision of the assay is measured as “within run” and “between run” and will
involve determining the coefficient of variation (CV) for results from 10 to 20
separate assays. A CV of 10% or less is generally acceptable.
f. Specificity: interference from other analytes (those commonly identified in
specific assays) should be determined by analyzing target analytes with and
without other drugs present.
g. Recovery or extraction efficiency is determined by analyzing a known amount of
analyte with and without extraction. The extraction efficiency will vary depending on
the type and condition of the matrix. Decomposed specimens will severely decrease
the analyst’s ability to extract drugs from a matrix. Use of an appropriate internal
standard should compensate for the variability in extraction efficiency. The best
practice is to obtain deuterated homologs of specific analytes for use as internal
standards. Laboratory resources may limit how many different deuterated internal
standards can be used, because they are expensive to acquire.
h. Stability of analytes is often overlooked or not performed for need to place the
assay in service. Knowing how long analytes will persist at the same concen-
tration in a specific biological matrix is an important criterion that ideally, at some
time, will be assessed.
i. Measure of uncertainty is a relatively new concept to forensic toxicology and is
being progressively implemented in laboratories across the country. The need
for determining uncertainty measurement for toxicology results was formally
realized during a suppression hearing in Washington State.25 Accreditation by
the ASCLD/LAB-International Program9 requires that uncertainty measures be
known for specific analyses. Uncertainty measurement is how one determines
the amount of variability that exists with a measured result, realizing that the true
uncertainty can never be known. To determine what the uncertainty is requires
identification of the most probable sources of uncertainty throughout the
analytical process, followed by the formulation of an uncertainty budget. As an
example, an uncertainty budget may be developed by considering the following:
 Reproducibility
 Purity of standards and reference materials
 Volumetric measurements as a function of maximum permissible errors and
temperature
 Variability of calibrator solutions with temperature corrections
 Variability of pipette calibration
 Variability internal standard/sample delivery
Other uncertainties that are less significant than those listed previously may include
 Storage conditions
 Instrument effects
 Sample/matrix effects
 Computational effects.
500 Wyman

The standard uncertainty is first calculated for each component of the budget. Different
calculations must be applied to different types of uncertainty. Guides for these
calculations are available.26 –30 Once standard uncertainties have been calculated,
the combined uncertainty is calculated by squaring the value of each standard
uncertainty, adding these together and then taking the square root of the sum of the
squared uncertainties. When calculating the combined uncertainty, if the individual
standard uncertainties for budget item is less than one-third of the maximum standard
uncertainty, it can be ignored.
The final uncertainty determination is made by calculating the expanded uncer-
tainty. This calculation requires a designated confidence interval of 95% or 99.7%
(two or three standard deviations). The confidence interval is represented by 2 or 3,
respectively, which is called the coverage factor “k.” The expanded uncertainty is
equal to the combined uncertainty multiplied by the coverage factor desired. The
ASCLD/LAB-International Program is considering requiring that uncertainty measure-
ment values be included on all toxicology reports, along with the measured value for
the analyte.
An excellent reference for further reading on quality assurance is provided by
Bramley and colleagues.30

POSTMORTEM INVESTIGATION

A strong forensic axiom is that no aspect of a death investigation is performed in

isolation. There is communication with all staff members through a morning “viewing
meeting” to discuss case history, medical records, pharmacy records, and the
circumstances of the death. An important consideration is that “results of toxicology
testing have to make sense” in light of the entire case findings. For example, GC/MS
analysis of specimens and identification of cocaine with no detectable cocaine
metabolites (benzoylecgonine, methyl ecgonine, cocaethylene) should give the toxi-
cologist pause, in that cocaine does not present by itself in postmortem specimens.
If this result occurs, the results of immunoassay screen for cocaine should be
examined and a repeat analysis should be considered. Another example is a vitreous
glucose greater than 600 mg/dL will often coincide with acetone in the blood and
vitreous volatile analysis, because acetone is a product of diabetic ketoacidosis. An
elevated glucose and acetone are complementary results. As is discussed later,
heavy lungs following an overdose of central nervous system (CNS) depressants is an
expected pathologic outcome.

Postmortem Interpretation of Toxicology Results

How does one determine that a drug concentration is therapeutic, toxic, or lethal?
Unlike experimental toxicology, forensic toxicology must rely on case reports pro-
vided in the literature and on the experience of the toxicologist/pathologist. Many
useful references are available to help guide the interpretation of postmortem drug
levels.31–36 An important aspect for interpreting toxicology results is knowledge of the
specific case history, the medical record, and record of prescribed (or unprescribed)
medications. Which forensic professional is responsible for drug interpretation will
vary depending on the coroner or medical examiner office. The pathologist may rely
on the toxicologist to provide the interpretation, or restrict the toxicologist to
providing only the identity and concentration of drugs. The best practice is for the
toxicologist and pathologist to have an open dialogue and collaborate on the
interpretation of toxicology findings. In addition to the drug concentration in post-
mortem specimens, other factors to be considered are drug-drug interactions, the site
 Forensic Toxicology 501

of specimen collection, date of collection, the route of administration, and the length
of time an individual has been taking a specific drug.

Signs of Toxic Pathology

Before toxicology testing begins, there may be evidence that a decedent was
exposed to certain toxicants based on findings at the external exam or from gross or
microscopic findings at autopsy. Examples include drugs in pockets of clothing, pill
fragments in the mouth or nose, powder around the nostrils, and transdermal patches
on the body or in the mouth. Examples of pathologic signs are a reddish hue to the
skin produced by carbon monoxide; hemorrhagic gastritis, which could have been
caused by heavy metals (arsenic or iron); cyanide, which also produces a rosy hue to
the skin and may be detected by smell by approximately 20% to 40 % of the
population at a threshold of .2 to 5 ppm37; Mees lines (white lines across the nail beds
of fingers and toes), which may be produced by arsenic and thallium38 along with a
hyperkeratosis of the palms of the hands and soles of the feet; Burton’s lines (blue or
black lines along the margin of the gum and teeth)39; signs of allergic reactions such as
a swollen tongue, rash, swollen airway, and voluminous lungs, which should prompt a
request for test for ␤-tryptase levels and serum immunoglobulin E; and fresh needle
punctures and/or old track marks of the skin, which suggest intravenous drug abuse.

Postmortem Redistribution
The propensity for certain drugs to have artifactually elevated concentrations in heart
blood following death is known as postmortem redistribution (PMR).40 – 42 Because
the drug concentrations are not representative of blood levels when the individual was
alive, use of drug concentrations in heart blood for interpretation can lead to
erroneous conclusions about the cause of death. As the time interval between death
and blood collection increases, the concentration of drug in heart blood increases.
Blood collection sites farthest from the central compartment organs will be least
affected by PMR. For purposes of interpretation, the preferential order of collection is
femoral, then iliac, then subclavian vessels, and then heart. Drug levels in blood from
the latter two collection sites are best left uninterpreted.
The phenomenon of PMR can be best characterized by thinking about drug
distribution when a person is alive. Certain drugs are sequestered by central
compartment organs (liver, lungs, and heart) at intracellular concentrations higher
than the circulating blood. Examples of drugs that exhibit high levels of PMR are
tricyclic antidepressants (eg, amitriptyline) and the analgesic propoxyphene. Ante-
mortem concentrations of these drugs may be greater than tenfold higher in liver
compared with circulating blood levels.43 To maintain this concentration gradient
during life requires energy and integrity of cell membranes. Cell death results in a loss
of energy (oxidative phosphorylation of adenosine triphosphate ceases) and cell
membranes are not maintained.

Determination of Dose in Postmortem Specimens is a Poor Practice

In clinical toxicology or therapeutic drug monitoring it is possible to calculate a dose
consumed based on blood concentration, body weight, and the volume of distribution
(Vd) of the drug as shown:

DOSE ⫽ Body Weight * Conc * Vd

In drug overdose cases it is natural to ask how many pills or capsules were
consumed by the decedent. If prescription bottles are available, this number may be
502 Wyman

determined by counting the pills left in the bottle and correlating the pills consumed
with how they were prescribed. To attempt to calculate dose after death based on
postmortem blood concentration is a poor practice44 for the following reasons:
1. Vd is never known for a specific individual and varies depending on body type
(body mass index).
2. Whether a drug concentration is at steady state is not known (is distribution
complete?).
3. PMR may elevate drug concentration from two- to tenfold.
4. Vd is based on plasma/serum concentrations, and postmortem toxicology is
performed on whole blood.
The fallacy of calculating pill counts should be pointed out to any pathologist who
has to respond to questions from family members and/or law enforcement. One
should never present postmortem pill counts as evidence in court.

Cases of Suspected Overdose

Confirmation of suspected drug overdose cases should be accomplished using
additional specimen analyses. If a lethal level of drug is determined for blood, then
analysis of liver will demonstrate whether the high level is a systemic intoxication. Not
following with a confirmation of systemic toxicity can sometimes lead to the wrong
conclusions, such as when an implantable drug delivery system (drug pump)
continues to release drug after death.

Tolerance or Loss of Tolerance

When evaluating drug levels in a deceased individual, it is important to review the
history and/or medical records to determine the degree of tolerance, if any, that might
have existed. Tolerance is an acquired phenomenon as a result of continued exposure
to a chemical substance. Mechanisms of tolerance may include (a) an increase in
metabolism of a drug as a result of metabolic enzyme induction and (b) desensitiza-
tion of the receptor with a decrease in pharmacologic response. Individuals in a
methadone maintenance program will have levels of methadone exceeding 1 mg/L,31
whereas naïve users can expire from blood levels less than .2 mg/L. Whether
tolerance to opiates, alcohol, or other drugs exists should be established before a
drug level is ruled an overdose. Conversely, a loss of tolerance (opiates) by persons
entering a drug rehabilitation program may result in their death if when they leave the
program they resume their addiction at the same level as before they entered
rehabilitation.

Conflicting Causes of Death in Litigated Cases

If the toxicologist’s report provides an interpretation of the drug level detected
(therapeutic, toxic, or lethal), it is always important to review the case history as to
how the individual died. Even though a high level of heroin or cocaine might be
considered lethal and a reasonable cause of death, reporting the drug as lethal could
pose a problem if the immediate cause of death was a gunshot wound to the head
and the case is a homicide. Reporting the drug level as lethal under these circum-
stances would provide the perpetrator a ready-made defense.

Specimen Collection in Decomposed Bodies

Depending on the state of decomposition, specimen collection from decomposed
bodies can be quite limited. The traditional specimens of blood and vitreous are the
 Forensic Toxicology 503

first fluids to be lost; if the temperature is warm, these matrices may be gone after 48
hours decomposition. Other available specimens to be collected may include liver,
kidney, muscle, bone, hair, nails, insect larvae, and/or soil beneath the remains. Liver,
the traditional matrix used for interpretation of drug levels when blood is not available,
should not be used for drug quantitation if the body is badly decomposed. After a few
days decomposition, drug levels in liver can increase several-fold higher than what
would have been measured at the time of death.45 This change is a result of anatomic
location (adjacent to the gastrointestinal tract) and fluid loss that decreases liver
mass. This dramatic increase in liver drug concentration can easily lead to erroneous
interpretation as to cause and manner of death. Drug concentration in muscle
changes much less over time than that of liver and may be a more reliable matrix for
interpretation.

Drug-Drug Interactions
Although there are many different categories of drug-drug interactions that one could
describe (such as drugs and dietary supplements or drugs and food), the three
principal interactions involve
1. Induction and inhibition of metabolism. Detoxification of drugs and chemicals in
the body is carried out by reactions known as phase I and phase II metabolism.
Phase I mostly involves oxidation and, to a lesser extent, reduction or hydrolysis of
substrates, whereas phase II concerns the conjugation of substrates with small
molecules such as glucuronide, glutathione, sulfate, glycine, acetate, and others.46
Induction or inhibition of metabolism of one drug by another is almost always a
result of phase I enzymes, principally hepatic cytochrome P-450 mixed function
oxidases. A large body of drugs function as substrates, inducers, and/or inhibitors
of this enzyme system.47 One drug inducing the metabolism of another drug, by
causing the production of more enzyme, may decrease the efficacy of the
metabolized drug. Conversely, inhibition of cytochrome P-450 metabolism by a
drug can cause an increase in toxicity of another drug. There are primarily five
families of cytochrome P-450 found in humans.48 Within these families, CYP3A4,
CYP2D6, CYP2C9, CYP2C19, CYP1A2, and CYP2E1 are of importance.46 Keep-
ing track of which drugs serve as substrates, inducers, and/or inhibitors for which
isozyme can be a daunting task. However, knowing that the vast majority (⬃ 75%)
of drugs are metabolized by only two isozymes, CYP3A4 and CYP2D6,46,49 helps
to simplify; also that ethanol, in addition to alcohol dehydrogenase, is metabolized
by CYP2E1. An in-depth discussion of cytochrome P-450 metabolism is outside
the scope of this article. Suffice it to say that interpretation of postmortem
toxicology results requires thinking about, and sometimes investigating the pos-
sibility of, induction or inhibition of metabolism of a drug by the presence of
another drug. For the most part, however, drug-drug interaction as a result of
induction or inhibition of metabolism is less of a concern with most drugs because
the change in the drug concentration is typically small, and most drugs generally
have a wide margin of safety and/or have multiple routes of metabolic elimination.
2. Disruption of drug storage reservoirs. The primary sites of drug storage are
plasma proteins (albumin and acidic glycoproteins) and tissue (organs, muscle,
and fat). If drugs bind to plasma proteins and/or tissues, they are no longer
available to cause their pharmacologic effect. When another drug with a greater
binding affinity for the binding site is introduced, the bound drug will become free
to act. An example of toxicity as a result of drug-drug interaction is when ibuprofen
is taken by a patient who is on warfarin. Use of ibuprofen (or other nonsteroidal
504 Wyman

antiinflammatory drugs) with warfarin is contraindicated because ibuprofen will

increase the concentration of warfarin and potentially cause unwanted hemor-
rhage.50 Therefore, some appreciation for the propensity of a drug to displace
another drug from plasma or tissue proteins can be gained by consulting the
percent protein binding data.31,36
3. Additive (synergistic) effects. The most frequent type of drug-drug interaction
that is encountered in postmortem, as well as human performance, toxicology is
increased toxicity because of additive or synergistic effects. A pharmacologic
effect common to numerous drug classes is CNS depression. CNS depression
occurs as a direct or indirect result of consuming alcohol, opiates, barbiturates,
benzodiazepines, tricyclic antidepressants, muscle relaxants, antihistamines, or
lithium. Within these drug classes are as many as a hundred different drugs, so the
likelihood of an individual consuming more than one CNS depressant is very
possible. Individuals dying from depressant polypharmacy experience CNS de-
pression, which leads to cardiac and respiratory depression (brain stem), followed
by pulmonary edema (lung weight increases from a norm of 350 g to ⬎1000 g) and
death. Evidence of pulmonary edema may be visible at the death scene in the form of
a “foam cone” on the nose and mouth. If a CNS depressant overdose is suspected as
the cause of death, autopsy should reveal edematous lungs weighing in excess of 500
g along with a full bladder. Without these findings, a conclusion that the person died
from an overdose of depressant drugs should be suspect.

Drug Polymorphisms and Pharmacogenetics (Pharmacogenomics)

Polymorphism means difference in phenotype between individuals, and pharmaco-
genetics is the study of the genetic variations that cause differences in the drug
response among individuals.46 These terms are used interchangeably, along with
descriptions of “fast” and “slow” metabolizers. One of the first isozymes to be
characterized was N-acetyltransferase 2, which causes the acetylation of isoniazid.
Other drugs that have been shown to have variable metabolism as a result altered
genetic makeup include succinylcholine (defects in pseudocholinesterase), nortripty-
line and codeine (CYP2D6), mephenytoin (CYP2C19), warfarin, phenytoin, tolbut-
amide (CYP2C9*3), midazolam (CYP3A5), nicotine (CYP2A61B), methadone, selegi-
line, propofol, efavirenz, and cyclophosphamide (CYP2B6) .

SUMMARY

This article is intended to provide the reader an overview of principles, proce-

dures, and practices in a modern forensic toxicology laboratory. Future trends in
forensic toxicology may include a change in analytical procedures where LC
tandem mass spectrometry, time of flight mass spectrometry, and/or capillary
electrophoresis are the standard analytical methods. Certification of analyst and
accreditation of laboratories will be a universal requirement. Laboratories will be
able to minimize the use of paper by using electronic documentation. The overall
effect of becoming a paperless laboratory will be an increase in efficiency,
decrease in case turnaround times, and a reduction in cost. Interpretation of
toxicology results from the perspective of pharmacogenetics will undoubtedly
affect decisions concerning the cause and manner of death, as well as in legal
proceedings. For further reading, an excellent introductory text, Principles of
Forensic Toxicology has been provided by Levine.51
 Forensic Toxicology 505

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