Forensic Pharmacology
Page 4
and each chemical can be released more or less easily than the
other from that material. Thus, there are two phases in a chro-
matographic system, a stationary phase to which the chemicals
adhere and a mobile phase that passes over the stationary phase
and takes with it the released chemical.
Gas chromatography (GC) uses a thin column made of stain-
less steel or glass. The stationary phase is a liquid such as methyl
silicone or a solid such as silica, and the mobile phase is a gas,
usually helium or nitrogen. As the mobile phase moves along the
stationary phase, volatile chemicals, depending on the heat of the
column, leave the stationary phase and travel in the mobile phase
to the end of the column, where a detector is located. Chemicals
with lesser affinity for the stationary phase are released before
those with greater affinity. As each chemical reaches the end, the
detector sends a signal to a recorder. The time it takes for a chemi-
cal to reach the detector from the time the sample is placed in the
column is termed the retention time. Chemicals are identified by
their retention time for a given separation system.
If liquid were used instead of gas for the mobile phase, this
procedure would be termed high-performance liquid chroma-
tography (HPLC or LC). Volatile chemicals are more easily sepa-
rated using GC, while chemicals in solution are separated using
HPLC (Figure 3.2).
30 Forensic Pharmacology
Figure 3.2 A scientist prepares a high-performance liquid
chromatography (HPLC) machine to analyze a blood sample. The
results of the test are visualized on the monitor to her right.
While retention time might be helpful in identifying a chemi-
cal, it may not be accurate enough, and an additional technique
must be applied to confirm the chemical’s identity. The gas and
chemical exit the GC and flow into an attached instrument called
a mass spectrometer (MS). Inside the MS, electrons or chemicals
bombard the chemical in the gas, resulting in its fragmentation
into smaller pieces of varying molecular weights. Here, each
chemical is broken down into various size fragments, with the
total group of fragments representing a specific chemical, much
like a fingerprint. Thus far, no two chemicals have produced the
same fragment pattern. The fragments pass through an electric
or magnetic field and are separated according to the mass of the
fragment. The spectrum of fragments is compared to thousands
Drug Analysis
31
of spectra in a library of chemicals and is identified. A known
amount of pure chemical is tested, and the results are then com-
pared with the unknown sample to be certain of the identifica-
tion and to allow quantification. The gas chromatography/mass
spectrometry (GC/MS) technique is very sensitive, and can
detect chemicals in the nanogram (ng) range. Results obtained
by GC/MS are considered confirmatory.
Screening and confirmatory tests have cutoff values. The val-
ues for drugs of abuse are provided in Table 3.1. These values are
based on various factors, including the precision and accuracy
of the individual test systems. If the test result is higher than the
cutoff value, the result is presumed positive; if it is lower, the
result is presumed negative. This does not mean that the drug
is totally absent, only that its concentration is below the cutoff
value. It may become important for a particular case to deter-
mine using other assays whether the drug is, in fact, present at
any level.
Thin layer chromatography (TLC) uses the same principles
as GC or HPLC but is performed on a glass plate containing an
adsorbent, such as silica or alumina, that attracts other molecules
to its surface. A small portion of the sample to be analyzed is
spotted on the plate. The plate is placed upright in a tank con-
taining a small amount of solvent that then rises up the plate and
separates the components of the sample. The separated compo-
nents can be located with an ultraviolet lamp or by spraying the
plate with chemicals to produce color.
Capillary electrophoresis, a relatively new technique, uses
an electric current to separate compounds based on their size,
charge, and mobile phase solubility. This technique requires
small amounts of sample. An analytical technique that provides
enhanced specificity and sensitivity for detection of chemicals is
LC/MS/MS. This technique separates compounds by HPLC and
then uses the MS to fragment the separated compounds. Unlike
32 Forensic Pharmacology
Table 3.1 Cutoff Values for Urine Drug Testsa
FEDERALb
NON-FEDERALc
Drug
Screeningd
Confirmatorye
Screening
Confirmatory
Cannabinoids
50
50
Δ9-tetrahydro-
cannabinol-9-COOHf
15
15
Benzoylecgonineg
300
150
300
150
Phencyclidine
25
25
25
25
Amphetamines
1,000
1,000
Opiates
2,000h
300
Morphine
2,000i
300
Codeine
2,000
300
Benzodiazepines
300
300
Barbiturates
300
300
Methadone
300
300
Methaqualone
75
75
Propoxyphene
300
300
Alcohol
0.02%
0.02%j
a All values are expressed as ng/ml except alcohol, which is expressed as grams/100 ml.
b DHHS mandatory standards for federal agencies monitor only for five major drugs of abuse. All laboratories are certified and use the same cutoff values as regulated by SAMHSA. See 49CFR40.87.
c Local nonregulated testing for law enforcement (driving while impaired) or random drug test (employment, parole, child custody, sports, drug rehabilitation). These values may differ among commercial laboratories; average values are presented.
d Testing by immunoassay.
e Testing by GC/MS.
f Metabolite of marijuana.
g Metabolite of cocaine.
h Federal standards were set higher to account for the possibility that poppy seed foods had been ingested.
i A morphine level of 2000 ng/ml or more requires a test for 6-monoacetylmorphine (6-MAM, heroin metabolite) with a cutoff at 10 ng/ml.
j Testing by gas chromatography.
Drug Analysis
33
single MS analysis, however, some fragments are selected and
then further fragmented.
Samples that contain volatile chemicals at room temperature
are analyzed differently. A closed container with blood at room
temperature will have volatile chemicals in the airspace above the
blood sample. A definite volume of air above the sample o
f blood is
drawn into a syringe and injected into a chromatograph. For each
volatile chemical, there is a definite ratio of the concentration of
chemical above the liquid phase relative to the concentration in
the liquid phase at a given temperature. (This principle is known
as Henry’s law.) Thus, determining the amount of chemical in the
sample taken above the liquid allows calculation of the amount in
the liquid. This technique, known as headspace gas chromatogra-
phy, is valuable for determining levels of ethyl alcohol, aldehydes,
ketones, petroleum distillates, halogenated hydrocarbons, and
gases such as nitrous oxide, methane, and freon.
DETERMINING BLOOD ALCOHOL CONCENTRATION
Blood alcohol concentration (BAC) is often based not on an
actual sample of blood but rather on the concentration of alco-
hol in a sample of breath (Figure 3.3). Alcohol is volatile, and,
as described by Henry’s law, there is a constant relationship
between the amount of alcohol vapor found in a volume of air
(breath sample) and the amount of alcohol found in a volume
of liquid (blood). All breath-testing equipment uses the blood-
breath ratio of 2,100:1 for alcohol. This means that the amount
of alcohol found in 2,100 milliliters of breath is equivalent to the
amount of alcohol found in 1 milliliters of blood.
This ratio may vary from individual to individual and, under
certain conditions, even within the same individual. Determina-
tion of a BAC from a breath sample may not always be accurate,
and this is often a point of argument in the courtroom.
34 Forensic Pharmacology
Figure 3.3 A breathalyzer measures the amount of infrared (IR)
energy absorbed by alcohol molecules. In this illustration, IR energy
from a lamp (1) travels through a chamber (2) holding the subject’s
breath. As the IR energy exits the chamber, it is focused by a lens (3),
passed through IR filters (4), and then converted into electrical signals
(5). A computer (6) receives the electrical signals and computes the
blood alcohol concentration.
The new automated breath-testing instruments use infrared
technology. Different chemicals absorb different amounts of
energy at different frequencies of the electromagnetic spectrum.
The electromagnetic spectrum ranges from large-wavelength
radio waves to small-wavelength gamma rays. Infrared radia-
tion, which is not visible to the human eye, has wavelengths
slightly longer than red, the last color of our visible rainbow.
At the other end of the rainbow is the color violet. Just below
violet, again not visible to the human eye, is ultraviolet. When a
breath sample is analyzed, the sample is irradiated with specific
infrared wavelengths, and the chemicals in the breath absorb
some of the energy. Based on the amount of energy transmitted
from one end of the instrument and the amount detected at the
other end of the instrument, one can determine the amount of
energy absorbed and thereby the concentrations of alcohol pres-
Drug Analysis
35
ent. Since other substances in the breath may also absorb energy,
although in amounts different than alcohol at the different
wavelengths, the instrument is calibrated at several wavelengths
to take into account these interfering substances.
Once a drug has been identified and its concentration deter-
mined, the forensic scientist might be able to form an opinion
as to whether a causal relationship exists between drug and the
incident under investigation. If the concentration is too low
to conclude causality, other explanations for the event may be
sought. The forensic scientist has available vast amounts of litera-
ture to assist in making this determination. The forensic scientist
Drug Recognition Experts
Alcohol is not the only chemical that causes one to become
impaired and drive erratical y. Any chemical that affects men-
tal functions, including some common prescription drugs as
well as controlled substances, can impair the ability to drive.
In the 1980s, a new program was instituted to certify police
officers as Drug Recognition Experts (DRE). DREs conduct a
12-step evaluation test that enables the officers to determine
whether an individual is under the influence of alcohol or other
drugs and determine the type of drug causing the impairment.
The 12 steps include a breath test for the presence of alcohol;
a discussion with the arresting officer; a preliminary exami-
nation of the eyes to determine pupil size and a measurement
of the pulse; an examination of the eyes for involuntary move-
ment, or nystagmus, and convergence; an evaluation of
the four psychophysical divided attention examinations (the
(continues)
36 Forensic Pharmacology
(continued from page 37)
one-leg stand test, the walk-and-turn test, the finger-to-nose
test, and the Romberg balance test); a measurement of blood
pressure, body temperature, and a second measure of the
pulse; a darkroom examination of pupil size and reaction; a
test of muscle tone; an examination of the skin for injection
sites; the noting of any statements made by the suspect;
the noting of the DRE’s opinion as to whether the suspect is
under the influence of drugs and, if so, which one; and, final y,
obtaining a blood or urine sample.2
The tests are designed to predict which of seven catego-
ries of drug the suspect may have used: (1) CNS depressants,
(2) CNS stimulants, (3) cannabinoids, (4) phencyclidine, (5)
opioids, (6) hallucinogens, and (7) inhalants. The combination
of results from the laboratory analysis of the blood or urine
sample and from the 12-step evaluation test will help decide
whether the defendant was impaired at the time of the stop.
can also add to the literature by publishing results from unusual
and interesting cases. Forensic scientists often belong to one or
more scientific associations and attend meetings where ideas are
exchanged and new information is presented.
There are two main groups that accredit forensic laboratories:
the governmental National Institute on Drug Abuse (NIDA) and
the nongovernmental American Society of Crime Laboratory
Directors (ASCLAD). Accreditation by the former is required for
the laboratory to perform workplace testing for federal agencies.
The groups monitor personnel training and development, record
Drug Analysis
37
keeping, evidence control, quality control, and, most impor-
tantly, proficiency testing, which helps ensure the accuracy of the
scientists and of the laboratory procedures.
SUMMARY
Drugs can be identified and their concentrations quantified
using a variety of techniques. Some of the techniques screen
the unknown sample to narrow the number of possible drug
categories. The techniques of chromatography and mass spec-
trometry are used routinely for identification and quantifica-
tion of chemical
s. Quantitation of drug in biological samples is
important to establish a causal relationship between drug and
effect. Various biological samples can be analyzed, but blood is
best for establishing causal relationships. Urine testing can indi-
cate prior use of the drug but has limited value in establishing
causality. A drug recognition expert (DRE) is trained to exam-
ine people and, based on a battery of tests, determine whether an
individual is under the influence of a particular drug.
4
Drug Abuse
and Teenager
Statistics
An individual might begin using drugs to diminish anxiety and
avoid dealing with problems, or to experience euphoria. Drugs
used for their euphoric effect are sometimes termed recreational
drugs. Use of such drugs often involves development of physical
and/or psychological dependence. Psychological dependence is
loss of control regarding drug use for either its positive effects or
to avoid negative effects when the drug is unavailable. For exam-
ple, an individual may make several unsuccessful attempts to
stop using drugs and/or spend much time and effort in obtain-
ing drugs. These are also signs of physical dependence, which
additionally involves developing tolerance, a decreased sensitiv-
ity to the drug, and exhibiting withdrawal symptoms if the drug
is not available.
Tolerance can develop in two ways. In pharmacokinetic toler-
ance, the drug is metabolized more quickly, thereby lowering the
blood levels. In pharmacodynamic tolerance, cells adapt to the
presence of the drug and are no longer affected at the usual concen-
tration. Either way, higher doses of the drug are required to achieve
a certain effect. When tolerance develops to pharmacologically
similar drugs, this is termed cross-tolerance, and one drug may
38
Drug Abuse and Teenager Statistics
39
substitute for another. Some drugs of abuse are more likely to
cause psychological dependence, while others cause both types
of dependence. The word addiction is sometimes used to describe
these states of dependence along with compulsive drug use.
If the user is drug dependent and does not get enough drug
to satisfy his or her craving, withdrawal begins to set in. With
some drugs, withdrawal is a very painful experience. Signs and
symptoms may include depression, aggression, restlessness, irri-