1404 TOXICOLOGY/Methods of Analysis ± Post Mortem
Methods of Analysis ± Post
Mortem
O H Drummer, Victorian Institute of Forensic Medicine,
Monash University, Melbourne, Australia
Copyright # 2000 Academic Press
doi:10.1006/rwfs.2000.0545
Introduction
Drug detection in postmortem specimens has many
uses, all of which assist the investigating authorities in
providing the relevant information pertaining to a
case (Table 1). Ultimately, toxicology testing results
will assist the coroner, medical examiner or equiva-
lent in establishing evidence of drug use, or by refut-
ing the use of relevant drugs. This latter is important
because a pathological examination will often not
show indicia suggestive of drug use. Drug use can
only be confirmed by appropriate toxicology testing
procedures. Clearly, in cases of sudden and unex-
pected death, use of drugs may provide a cause of
death, or at least provide evidence of drug misuse or
drug abuse. (`Drug misuse' is used here to define cases
where inappropriate doses or drug combinations have
been used, whereas `drug abuse' is restricted to delib-
erate and usually recreational use of drugs of abuse.
Neither term necessarily implies suicidal intent.)
Toxicology testing is particularly important in
victims of homicide, to whom drugs may have been
given by the assailant to reduce consciousness, and in
cases in which drugs were used by the victim. In this
latter scenario, modification of behavior by drug use
may be important in criminal trials, not necessarily to
mitigate the intent by the accused, but primarily to
reconstruct, as far as possible, the events that led to
the act. Such reconstruction may involve corrobora-
tion of witness accounts of drug-using behavior.
Typical drugs used in these cases are alcohol,
amphetamines, cocaine or one of the benzodiazepines
(alprazolam, diazepam, flunitrazepam, etc.). Victims
or perpretrators of violent crime may also have con-
sumed medication to treat a psychiatric problem or a
host of other medical conditions. The presence of
Table 1 Reasons for drug testing in postmortem cases.
Establishing drug use in victims of homicide
Establishing drug use in drivers of motor vehicles
Establishing drug use in persons involved in workplace accidents
Establishing drug use in other cases of sudden and unexpected
death
Assisting investigators with estimation of timing of drug ingestion
drugs may therefore be indicative of such treatment,
or at least confirm the person concerned has taken the
medication. In some cases these medications may
even have contributed to behavioral problems.
In practice, deceased persons have often consumed
two or more drugs, and in many cases the investigating
authority (pathologist, coroner, etc.) is not aware of all
the drugs being used. Since the great majority of cases
(470%) involve more than one drug, it is advisable to
conduct a broad drug screen to include most of the
common drugs, rather than target the analysis to one
or a limited range of drugs suggested by the circum-
stances. This will also allow experts to determine if any
adverse drug interactions have occurred.
Specimens
The preferred specimens collected at postmortem will
depend on the type of case. Most typically, one or
more blood specimens and urine are collected, al-
though as Table 2 illustrates, a number of other
specimens should be taken in certain case types. A
useful forensic technical procedure in the autopsy
suite is to take a `full' set of specimens in all but the
most obvious natural cause investigations. This will
avoid the embarrassment of collecting insufficient or
inappropriate specimens and give the toxicologist the
best chance to complete the analytical investigation
satisfactorily.
Blood is the most useful specimen that can be
collected because drugs present in this fluid can best
be related to a physiological effect and can be used to
assess the likelihood of recent drug use or exposure to
chemicals. A number of problems are associated with
the collection of this fluid in cadavers. Two 10 ml
samples of blood are recommended, one to be used
for blood alcohol analyses and the other for blood
toxicology. The splitting of the two blood specimens
reduces the possibility of contamination in the la-
boratory and enables the blood alcohol specimen to
be retained separately from the other blood specimen.
Forensic technicians or pathologists should be aware
that the collection of peripheral blood reduces the
possibility of postmortem artifacts frustrating inter-
pretation of any positive results (see Redistribution).
The preferred collection site is the femoral region
(leg); however, failure to ligate the femoral vessels
can allow `contaminated' abdominal blood to be
withdrawn. Autopsy procedures should therefore
accomodate these problems. Drug content is reported
as milligrams per liter.
Urine is the second most important specimen col-
lected. Since concentrations of drugs and metabolites
of drugs are usually much higher than in blood, urine
 TOXICOLOGY/Methods of Analysis ± Post Mortem
Table 2 Recommended minimum specimens to be collected post mortem.
Type of case
All cases
Homicides and suspicious cases
Drug-related cases
Volatile substance abuse cases
Biochemical abnormalities (insulin, etc.)
Heavy metal poisoning
provides a valuable specimen for assessing drug use
over the previous day or two. Urine can be collected
after the opening of the abdomen, or by direct punc-
ture of the bladder. An autopsy is therefore not
necessary for collection of this specimen, or even
blood and vitreous humor (see later). Drug content
is normally reported as milligrams per liter.
Vitreous humor is an ideal fluid to collect with
`positive' blood because the alcohol content of
vitreous humor is very similar to that of blood and
can prove useful in excluding putrefactive formation
of alcohol in blood and visceral contamination. Vit-
reous humor is also a useful fluid for a range of drugs,
including digoxin and antidepressants, as well as a
number of biochemical markers. Since vitreous
humor can be easily collected, it is strongly recom-
mended that this specimen is included in a routine,
sudden death investigation. Drug content is normally
reported as milligrams per liter.
The liver is traditionally a favored tissue for toxi-
cologists because drugs are often found in higher
concentrations there than in blood, it can be readily
homogenized and, of course, it is the main metabolic
organ. A liver sample should be collected in all cases
of suspected drug use. A 100 g aliquot is sufficient for
most analyses. The right lobe is preferred, as this is
least subject to postmortem diffusion of drug from
bowel contents and the mesenteric circulation (see
Redistribution). Drug content is normally reported as
milligrams per kilogram of wet weight tissue.
Gastric content is invaluable in cases of suspected
poisoning. The aim of using this specimen is to es-
tablish the actual content of drug (or poison) remain-
ing in this organ at death, and it may allow the route
of drug administration to be determined. Drug resi-
dues can be isolated by direct extraction with metha-
nol, or another solvent, and analyzed by conventional
chromatographic techniques. When little or no fluid
is present in the stomach, provision of the whole
stomach allows the analyst to dissolve any drug ad-
hering to the sides of the walls. Toxicologists should
be aware that small quantities of drug will derive
1405
Recommended specimens collected
Peripheral blood (2 6 10 ml)
Urine (10 ml)
Vitreous humor (2±5 ml)
Liver, bile
Gastric contents, bile, liver
Lung fluid or tied-off lung, liver
Serum
Liver, hair, kidney
from the bile, especially during agonal processes,
hence drug content in the stomach does not neces-
sarily imply oral ingestion. Results should be reported
as milligrams (total gastric content).
Lung fluid, or tied-off lungs, are recommended in
cases of suspected volatile substance abuse. Since
quantitative results are rarely interpretable, `detected'
or `not detected' results alone are usually sufficient
(Table 3).
Bile is a useful fluid for morphine/heroin detection
because biliary concentrations are much higher than
in blood. A number of other drugs are also found in
bile in relatively high (and therefore more easily de-
tectable) concentrations, including other narcotics,
benzodiazepines and glucuronide metabolites. Report
bile results as milligrams per liter.
Occasionally other specimens can provide valuable
information in a case. Hair can provide a history of
drug use, or exposure to chemicals if chronic expo-
sure is thought to occur. Hair can therefore provide
evidence of drug use for much longer periods of time
than urine. The relation between dose and hair con-
centration is usually poor, although some compari-
sons can be made as to the extent of drug use, e.g.
regularity of heroin use. Hair concentrations are nor-
mally reported as micrograms per gram weight.
Table 3 Useful substance detections in various tissues.
Tissue Substances detected
Blood/urine/liver/hair/ All drugs and poisons
gastric contents
Vitreous humor Alcohol, antidepressants, narcotics,
creatinine, urea, glucose
(410 mmol l71)
Bile Morphine and other narcotics,
benzodiazepines
Lungs Volatile substances (toluene and
other solvents, butane and other
aerosol gases, automobile and
aviation fuels)
1406 TOXICOLOGY/Methods of Analysis ± Post Mortem
Samples of brain tissue may be more relevant for
some centrally active (i.e. in the brain and spinal cord)
drugs, such as morphine; and skin (with associated
subcutaneous tissue) may show large deposits of drugs
left behind after an injection. Results are normally
expressed as milligrams per gram wet weight tissue.
In cases of extreme putrefaction, the recommended
list of specimens will no longer be appropriate. Mus-
cle tissue, hair and bone can be useful specimens in
this type of case, although the physical state of the
body will determine what specimens are available for
collection. Body fluids will be present in some putre-
fied bodies but this is no longer blood, rather liquified
tissues; but this fluid can be used to screen for the
presence of drugs. Quantitative results are of little use
in badly putrefied cases.
General Techniques
The range of techniques available for the detection of
drugs in the specimens collected post mortem are
essentially identical to those collected ante mortem.
These range from commercial kit-based immuno-
assays (enzyme multiplied immunoassay technique
(EMIT), fluorescence polarization immunoassay
(FPIA), cloned enzyme donor immunoassay
(CEDIA), radioimmunoassay (RIA), etc.), traditional
thin-layer chromatography (TLC), to instrumental
separation techniques such as high-performance liq-
uid chromatography (HPLC), gas chromatography
(GC) and capillary electrophoresis (CE). Mass spec-
trometry (MS) is the definitive technique for establish-
ing proof of structure of an unknown substance and
can be linked to GC, HPLC and more recently to CE.
The specimens analyzed in postmortem cases are
most often blood and liver, rather than urine and
serum, which are used in antemortem analysis, and
the other specimens listed earlier. The use of blood
and liver, and indeed all other postmortem speci-
mens, require separate validation to those methods
used in antemortem analysis. The methods used re-
quire modification to ensure a reliable extraction
recovery, a low level of interference and reproducible
quantitative results. Special attention to these factors
is required on partly or fully putrefied specimens to
ensure lack of interference from endogenous sub-
stances. Cutoff values often used in workplace, sports
and drugs-of-abuse testing are no longer appropriate
in postmortem cases involving specimens other than
urine. Even postmortem urine should not normally be
tested to cutoff limits used in drugs-of-abuse testing
because the presence of a small concentration of drug
may be of forensic significance.
The range of immunoassays used in antemortem
analysis can also be used in postmortem analysis,
providing suitable modification of the preparation
of the specimen is conducted. Urine-based kits can
be used for urinalysis; however, blood or tissue
homogenates require special treatment to remove
matrix effects. Urine is often unavailable in pos-
mortem cases. Precipitation of blood proteins by
treatment with methanol, acetonitrile, dimethylform-
amide or acetone, and direct analysis of the super-
natant are frequently used techniques; however, the
high-potency drugs are not always detected. Prior
extraction of blood or liver homogenates with a sol-
vent (e.g. butyl chloride) provides an improved
detectability, as a concentration step can be employed
and most interference has been removed. Not all
drugs are extracted with all of these techniques. In-
dividual validation must be conducted to ensure ad-
equate detectability for target drugs and to determine
the range of drugs that can, and those that cannot, be
detected.
False-positive results with immunoassays occur,
either from structurally related drugs, or from meta-
bolites of other drugs that are recognized by the
antibodies. While HPLC and GC techniques are
more specific than immunoassays, any positive result
should be confirmed by mass spectral identification,
unless sufficient validation of another method has
been conducted to assure courts of the reliability of
the result. Unconfirmed drug results, if reported,
should be flagged as presumptive, or words with
similar intent.
Solid-phase extraction, using small columns to ab-
sorb drug selectively from the matrix (e.g. Extrelut,
Sep-Pak, Bond-Elut, etc.), provides an excellent alter-
native to conventional liquid±liquid extraction tech-
niques. Solid-phase techniques have been published
for most analytes, tend to be quick, often provide
clean extracts and can be readily automated.
The use of deuterated internal standards provides
an ideal way to monitor changes in chromatographic
performance, and, most importantly, essentially elim-
inates matrix effects caused by poor recoveries of
drug. While recoveries of drug may vary from one
matrix to another, and even between calibrators, the
deuterated internal standard will correct for this. For
this reason, assays involving MS should use deuter-
ated internal standards wherever possible in post-
mortem analyses.
Recommended Techniques for
Postmortem Analysis
As indicated before, it is important that a drug screen
encompass the widest number of drugs and poisons
without seriously compromising the ability of the
 TOXICOLOGY/Methods of Analysis ± Post Mortem 1407

laboratory to work on sufficient cases. Urinanalysis
(or blood or another fluid) using one of the commer-
cial immunoassays, or even TLC, is recommended
for the main classes of drugs. These usually include
amphetamines, barbiturates, benzodiazepines, can-
nabinoids, cocaine metabolites and morphine-like
opiates.
In addition, a series of other (usually chromato-
graphic) tests is strongly recommended. The schema
shown in Fig. 1 illustrates a typical analytical profile
for routine case screening of blood. Blood is analyzed
for alcohol and is subject to a few screening techni-
ques aimed at capturing a wide selection of chemical
substances to which humans are likely to be exposed.
An acidic screen includes the nonnarcotic anal-
gesics (acetaminophen, aspirin), the nonsteroidal
antiinflammatory drugs (naproxen, ketoprofen,
ibuprofen, etc.), many of the diuretics (frusemide,
hydrochlorothiazide, etc.), the anticonvulsants (car-
bamazepine, lamotrigine, phenobarbital, phenytoin,
valproate), barbiturates and the more potent benzo-
diazepines, the xanthines, theophylline and caffeine.
The use of a solvent extraction technique at acidic
pH, or simple precipitation of blood proteins with
acetonitrile, enables these substances to be detected
by gradient HPLC with multiwavelength or photo-
diode array detection.
A basic extraction procedure using butyl chloride
(preferred solvent, but others are also suitable), or a
solid-phase extraction procedure using octadecyl-
bonded cartridges or mixed-phase cartridges, will
provide a reasonably clean extract from postmortem
blood (and other tissues) for analysis by capillary GC.
The use of an MS detector is preferred (to allow
simultaneous detection and confirmation), although
a nitrogen±phosphorus detector (NPD) will provide a

Figure 1 Extraction steps for blood analyses and substance classes likely to be detected. GC, gas chromatography; GC-MS, GC
mass spectrometry; HPLC, high-performance liquid chromatography.
1408 TOXICOLOGY/Methods of Analysis ± Post Mortem

higher sensitivity for many substances than full-scan Table 4 Likely extent of postmortem redistribution for selected
drugs.
MS. Electron capture detectors (ECD) are extremely
useful for benzodiazepines. The use of dual detectors Drug/drug class Redistributiona
(NPD and MS, or NPD and ECD) provide an addi-
tional degree of specificity and detection over one Acetaminophen Low
detector alone. Alcohol (ethanol) Low
These two screening procedures will also enable a Barbiturates Low to moderate
Benzodiazepines Low to moderate
number of unusual poisons to be detected. Organo-
Cocaine Low
phosphates and strychnine are readily detected by Digoxin Very high
GC-NPD, while HPLC of acid extracts enables de- Methadone Moderate
tection of a number of herbicides and other agricul- Morphine Low
tural chemicals. If circumstances suggest volatile Phenothiazines Moderate to high
Propoxyphene Very high
substance abuse, exposure to heavy metals, lysergic
Salicylate Low
acid diethylamide (LSD) and other nonamphetamine Serotonin reuptake inhibitors Low to moderate
hallucinogens, or other noxious substances not cov- Tricyclic antidepressants High
ered earlier, specific additional tests will need to be
performed. It is advisable to perform a blood test for a
Low, up to 20% elevation; moderate, 21±50%; high, 50±200%; very high,
morphine if heroin or morphine use is suspected (or 4200%.

needs to be ruled out) and the urine test for opiates is

negative, as morphine does not chromatograph well
underivatized. Heroin deaths have been missed if
screening for morphine is restricted to urine because
acute deaths in naive users may not show morphine in
urine.
Postmortem Artifacts in Analysis
Dying imparts a number of special processes that
affect the collection and analysis of specimens
obtained at autopsy.
advisable to reduce these processes by collecting
blood specimens as soon as possible after death
from the femoral region, with blood vessels tied off
to reduce contamination.
These processes are not limited to blood. Liver and
lung tissue show differences in the concentration of
drugs, depending on the nature of the drug and
whether diffusion of drug has occurred from neigh-
boring tissues or the blood supply. For example, the
left lobe of the liver is more likely to exhibit elevated
drug concentrations than the right lobe.

Redistribution Metabolism and bioconversion

Foremost is the process of redistribution, which
affects all analyses in which concentrations of drugs
in blood and tissues alter due to disruption of cellular
membranes, causing alterations of drug concentra-
tions within tissue elements and diffusion from one
tissue to another. This process is particularly signifi-
cant for drugs with high lipid solubility, as these
drugs tend to show concentration differences in tis-
sues and blood. Table 4 shows the extent of these
changes for selected drugs when comparisons are
made between blood collected from the heart and
that collected from the femoral region.
The femoral blood is least subject to redistribution
after death; however, drugs with much higher con-
centrations in muscle tissue will still diffuse through
the vessel walls and elevate the neighboring blood
concentrations. If the femoral vessels are not tied off
from the vena cava and aorta, then the process of
drawing blood can also extract blood from the ab-
dominal cavity that has been contaminated from dif-
fusion of gastric and intestinal contents. It is therefore
A number of drugs can undergo chemical changes in
the body after death. These chemical changes can be
either metabolically mediated or caused by sponta-
neous degradative processes. For example, the me-
tabolism of heroin to morphine occurs in life and also
occurs in recently deceased persons by the action of
blood and liver esterases. For this reason, heroin or
the intermediate 6-monoacetyl morphine are rarely
detected in cadaveric tissues. Morphine is therefore
the target drug. Aspirin is converted rapidly to salicy-
late by hydrolytic mechanisms. Most prodrugs acti-
vated by de-esterification or hydrolysis will be subject
to similar processes.
Nitro-containing drugs, such as the benzodiaze-
pines, nitrazepam, clonazepam, nimetazepam, fluni-
trazepam and others, are also rapidly biotransformed
after death to their respective amino metabolites by
the action of certain types of bacteria (obligate
anaerobes). Toxicologists must therefore target their
analyses to these transformation products rather than
the parent drug.
 TOXICOLOGY/Methods of Analysis ± Post Mortem
Sulfur-containing drugs, such as dothiepin, thio-
pental, thioridazine, etc., are also subject to bacterial
attack post mortem, leading to progressive losses
over time during putrefaction. Of course, the parallel
process of tissue loss will also affect the tissue con-
centration during putrefaction.
Chemical instability occurs in a number of drugs
and metabolites even when specimens are stored
frozen at 7208C. Some benzodiazepines and benzo-
diazepine metabolites, antipsychotics such as thior-
idazine, and the b-stimulant fenoterol show time-
dependent losses. Stability characteristics have not
yet been evaluated for many drugs.
Alcohol will be lost to evaporation unless sealed
tubes are stored at 7608C, however, alcohol can also
be produced by bacterial action on glucose and other
sugars found in blood. The use of potassium fluoride
as preservative (minimum 1% w/v) is required to
prevent bacterial activity for up to 1 month after
collection, when stored at 48C.
Reports
Once an analysis is complete, a report that accurately
details the analytical findings must be issued to the
client(s). These results should indicate the type of tests
conducted, the analytical method used (i.e. HPLC,
GC±MS, etc.), on which specimens the analyses were
conducted, and, of course, the result(s). The result(s)
should be unambiguous, using such terms as
`detected' or `not detected'. The use of the term `not
present' should be avoided, as it implys no possiblity
of the substance being present. A toxicologist can
rarely be so definite and can only indicate that a
substance was not detected at a certain threshold
concentration. A detection limit should therefore be
provided when tests for specific substances produce
`not detected' results.
For quantitative results, consistency in units is ad-
vised and should not be given with more significant
digits than the accuracy will allow. For example,
there is no point in reporting a result for blood mor-
phine as 0.162 mg l71 when the accuracy and preci-
sion of the method is +20%. A result of 0.16 mg l71
would suffice.
For drug screening results, it is advisable to provide
clients with an indication of the range of substances a
method is capable of detecting, and some indication
of the detection limits, i.e. `at least therapeutic con-
centrations' or `only supratherapeutic concentra-
tions'. Positive immunoassay results should also be
reported even if this presumptive detection has not
been confirmed. This information can be useful be-
cause it may imply (to an expert later investigating the
case) that the substance may have been present but at
1409
very low concentrations, or there was another immu-
noreactive compound which was not excluded in the
confirmation assay. To exlude these results could be
construed by courts as a deliberate withholding of
evidence.
To enable proper interpretation of evidence, all
reports should indicate the site of blood sampling,
and provide, where relevant, some comment on the
possibility of postmortem artifacts, such as redistri-
bution. By incorporating these comments, unin-
formed persons reading the report are less likely to
unwittingly misinterpret the results.
See also: Toxicology: Methods of Analysis ± Ante
Mortem.
Further Reading
Backer DR (ed.) (1995) Capillary Electrophoresis. New
York: Wiley.
Davies G (ed.) (1986) Forensic Science, 2nd edn Washing-
ton, DC: American Chemical Society.
De Zeeuw RA (1989) Modern chromatographic proce-
dures in systematic toxicological analysis. Journal of
Chromatography 488:199±213.
De Zeeuw RA (1995) Hair Analysis in Forensic Toxi-
cology. Abu Dhabi: Directorate of Police.
DFG/TIAFT (1992) Thin-layer Chromatographic Rf-
values of Toxicologically Relevant Substances in
Standardized Systems. Weinheim: VCH.
DFG/TIAFT (1992) Gas-chromatographic Retention
Indices of Toxicologically Relevant Substances on
Packed or Capillary Columns with Dimethylsilicone
Stationary Phases. Weinheim: VCH.
Drummer OH (1998) Adverse drug reactions. In: Selby H
(ed.) The Inquest Handbook, Leichhardt, NSW: Fed-
eration Press.
Drummer OH (1999) Review: Chromotographic screening
techniques in systematic toxicological analysis. Journal
of Chromatography 733:27±45.
Ghosh MK (1992) HPLC Methods on Drug Analysis. New
York: Springer.
Li SFY (ed.) (1986) Capillary Electrophoresis, Principles,
Practice and Applications. Amsterdam: Elsevier.
Maurer HH (1992) Systematic toxicological analysis of
drugs and their metabolites by gas chromatography±
mass spectrometry. Journal of Chromatography 118:3±
42.
Moffatt AC (ed.) (1986) Clarke's Isolation and Identifica-
tion of Drugs. London: Pharmaceutical Press.
Pfleger K, Maurer HH and Weber G (1992) Mass Spectral
and GC Data of Drugs, Poisons and their Metabolites.
Weinheim: VCH.
United Nations (1997) Recommended Methods for the
Detection and Assay of Barbiturates and Benzodiaze-
pines in Biological Specimens. Publication ST/NAR/28.
New York: United Nations.