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Systematic Drug Identification
N N Daéid, University of Strathclyde, Glasgow, UK
& 2005, Elsevier Ltd. All Rights Reserved.
Introduction
One of the main activities in a forensic chemical
laboratory is the examination of powders, pills, and
plant materials thought to be illicit or controlled
substances. The analysis of suspected drugs of abuse
occurs over a number of stages. These involve the
preparation of the analytical space within the labo-
ratory, a physical examination of the item including
its packaging, the use of a sampling protocol depen-
ding on the nature of the samples presented and the
analysis required and the analysis of the sample.
There are various analytical techniques commonly
used in the forensic examination of suspected drugs
and these include presumptive testing (color/spot
tests), thin layer chromatography (TLC), liquid chro-
matography (LC), gas chromatography (GC) with
either flame ionization detection (FID) or mass spect-
rometry (MS), Fourier transform infrared spectro-
scopy (FTIR), and ultraviolet (UV) spectroscopy. In
many situations the data obtained from these anal-
yses would then be compared with laboratory refer-
ence standards and/or a standard reference text such
as Clarke’s analysis of drugs and poisons.
The choice of technique which is used, depends
upon the requirements of the analysis and specifically
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Classification and identification of color photocopiers by
FT-IR and GC/MS. Journal of Forensic Sciences 43(2):
353–361.
Spence LD, Francis RB, and Tinggi U (2002) Comparison
of the elemental composition of office document paper:
evidence in a homicide case. Journal of Forensic Sciences
47(3): 648–651.
Tebbett IR (1991) Chromatographic analysis of inks for
forensic science applications. Forensic Science Review
3(2): 71–82.
Totty RN (1990) Analysis and differentiation of photocopy
toners. Forensic Science Review 2(1): 1–23.
Zlotnick JA and Smith FP (1999) Chromatographic and
electrophoretic approaches in ink analysis (Review).
Journal of Chromatography B 733(1–2): 265–272.
the questions and hypothesis to be addressed. Spe-
cifically this commonly includes:
* dmac_aq>p0015Determination of the identifica-
tion or confirmation of the presence of a control-
led substance in a sample;
* qmac_aq>p0020Quantification of any controlled
substance (i.e., determining the amount of the
controlled substance present); and
* determination of links between samples or syn-
thetic route used in sample production through
chemical profiling or characterization of synthetic
by-products and/or reaction impurities within the
sample.
Legal Aspects of Illicit Drug Analysis
The United Nations through their office on drugs and
crime (UNODC) has produced over the years three
major international drug control treaties, which are
mutually supportive and complementary. In addition
to including general provisions on illicit trafficking
and drug abuse, they seek to ensure the availability of
narcotic drugs and psychotropic substances for med-
ical and scientific purposes and to prevent their
diversion into illicit channels.
The treaties are:
* Single Convention on Narcotic Drugs, 1961 as
amended by the 1971 Protocol concerning
psychotropic compounds.
472 FORENSIC SCIENCES / Systematic Drug Identification
* United Nations Convention against the Illicit
Traffic in Narcotic Drugs and Psychotropic Sub-
stances, 1988.
The UNODC’s Legal Advisory Programme pro-
motes adoption and practical implementation of
these conventions. It addresses the needs of States
that have outdated drug control legislation, are most
vulnerable to criminal activity, or lack success in
major prosecution and asset forfeiture casework. The
program advises states on the drafting, adoption, and
application of all necessary legislation.
Each country generally has its own drug legislat-
ion, which in general has undergone some evolution
over the years. A comprehensive list of the different
legislative controls are to be found on the United
Nations web site at the following URL: http://
www.unodc.org/unodc/legal library/index-coun-
tries.html.
Laboratory Examination
Preparation of the Analytical Space
One of the most important (and fundamental) parts
of the examination of samples suspected of contain-
ing controlled substances is the initial preparation of
the space where the analysis is to take place. Benches
should be washed down with detergent, dried, and
then swabbed with methanol or similar solvent. The
swabs should be retained for analysis if required.
Fresh bench covering should be laid on the bench.
Sample packages should be opened individually and
the bench should be cleaned as described, in between
subsequent packages being opened. There should be
frequent changes of gloves and meticulous notes
require to be kept on all procedures undertaken.
Physical Examination
Packaging
Once drug samples are presented for examination,
their packaging should be checked to ensure that it is
intact. Any breech of the packaging should be re-
ported and a decision taken as to whether the ana-
lysis of that item should be continued. Packaging
materials for drug samples come in a variety of
forms, from wrapping materials such as cling film
(often around cannabis resin), tin, or aluminum foil
(powder samples and some tablets) to larger polye-
thene bags ranging from bank bags to bigger ziplock
type bags. In some cases linkages can be made from
sample to sample based upon the packaging materi-
als present and it is well worth spending time on their
examination. For example, cling film can be physi-
cally linked using polarized light techniques and
chemically linked using FTIR.
Samples
The physical description of an item, as well as the
types of analysis undertaken, will depend upon
whether that item is considered a bulk or trace sam-
ple. Each item should be described fully (with
diagrams if appropriate) including a description of
color, smell, and any packaging materials, which may
be present. If there are logos (ecstasy tablets) or marks
on the items (blocks of resin or packages of drugs),
these should be fully described and possibly pho-
tographed. Microscopic examination may also be
used to examine the morphological characteristics of
the material. This includes descriptions of different
types of crystals or other solids, which may be
present, the structure of tablets, the presence of dif-
ferent botanical features in seizures of plant materials.
Trace versus bulk samples A trace sample is con-
sidered to be one, which is barely visible to the naked
eye. Samples such as this will be present on the insides
of reaction vessels, scales, knives, and other drug par-
aphernalia. In many cases, trace samples are swabbed
from these items and are analyzed using confirmatory
techniques first. Such samples are very easily contam-
inated and significant care should be taken in their
analysis. Bulk samples require a sampling method-
ology and are generally subjected to presumptive tests
and TLC followed by confirmatory tests.
Sampling Protocols
Many laboratories have developed their own sampling
protocol for drug samples. In all cases, samples should
be taken from items, which have the same morphology
(also called sampling by attributes). This means that if
a seizure is found to contain different types of mate-
rials, then each type should be sampled separately. This
is particularly true for samples containing tablets with
different logos or of different colors, or items, which
have been packaged separately from each other. Where
blocks of resin are being sampled, this should be away
from the edges of the block as such edges can poten-
tially be used to make physical fits between the blocks.
In cases where samples of powder are seized, it is nec-
essary to ensure that the powder is homogenous which
is generally accomplished using standard techniques.
Two things generally dictate sampling protocols:
* A legal obligation which may require that all items
in a seizure are described and sampled.
 FORENSIC SCIENCES / Systematic Drug Identification
* A policy left to the expert, which will include a
description of the items and the selection of a
sample from the items.
The criteria for selecting the type of sampling pro-
tocol undertaken should include a balance between
the loss of completeness versus time saving.
In actual fact most sampling protocols are based
upon a mathematical distribution called the hyper-
geometric distribution (eqn [1]). Some laboratories
have adopted this as their sampling policy and will
only analyze a maximum number of samples or
packages from any one seizure regardless of the total
seizure size. The number analyzed varies depending
on the level of accuracy (99%, 95%, etc.) which is
acceptable.
ðM Cm ÞðN
M Cn
m Þ
Pðm=n; M; NÞ ¼ NC
½1
n
where
a!
a
Cb ¼
ða
bÞ!ðb!Þ
and n is the size of the subpopulation analyzed, m a
desired outcome, N the size of the sample, and M the
number of positive outcomes in the sample.
An international system of sampling, which has
been adopted and promoted by the UNDCP is as
follows.
Powders and Tablets and Packages
 Single package of material – The material should
be removed from all packaging and weighed to a
constant dry weight. The sample is then homogeni-
zed and a sample removed.
 More than one package – The material in each
package should be examined by eye for color dif-
ferences. The contents of each package should be
weighed (cleaning balance in between each weigh-
ing) and each package should be tested using a col-
or spot test or TLC. If it is assumed that all
packages contain the same material then;
if there are less that 10 packages all should be
tested
if there are between 10 and 100 packages 10
should be tested at random
if there are more than 100 packages the square
root of the total should be tested at random.
Liquids
If all of one phase then a sample of the liquid is
removed for testing. If there are more than one phase
473
present then a sample of each phase should be
removed for testing.
Identification and Quantification
Techniques
Presumptive Tests – Identification of Drug Class
The first tests carried out on a sample are pre-
sumptive tests to give an indication of the class of
drug which may be present in the sample. These are
generally performed on clean porcelain tiles, in so-
lution or on adsorbent substrates. The most common
type of presumptive test involves the addition of var-
ious reagents to the sample to produce a color. Other
tests involve the use of microscopy to identify tric-
homes in suspected cannabis samples or microcrys-
talline tests where the formation of specific crystals is
indicative of the class of drug present. In all cases
appropriate positive and negative control samples
must be used. Table 1 lists some of the results ob-
tained with different spot test reagents for common
drugs of abuse.
Advantages and disadvantages of presumptive
tests Most presumptive tests are carried out on
porcelain tiles. There are a number of clear advan-
tages to tests of this nature: they are cheap, quick,
and easy to use. They also have the advantage
of being portable if required and various ‘road side’
test kits have been developed on the basis of color
tests.
The main disadvantages are that the colors formed
are subjective, may change over time, and may be
produced by more than one drug compound or by
compounds other than controlled substances. These
tests are also of limited sensitivity (few micrograms)
and so are often not suitable for the analysis of trace
samples.
Thin Layer Chromatography – Identification of
Drugs within a Class
Having tentatively identified the class or classes to
which the drug belongs to using presumptive testing,
the next stage in drug identification is to examine
which types of drugs from the class are present in the
seizure. This can be achieved using TLC, a technique
which has the advantages of being both rapid and
cheap. The separation process depends on the re-
lative strength of interaction of the sample compo-
nents with a stationary phase, and a solvent moving
through this material, the mobile phase. The solvent
in which the sample is dissolved must be one that is
suitable for the analysis of the suspected compound.
474 FORENSIC SCIENCES / Systematic Drug Identification

Psilocin psilocybn
Suitable solvents Suitable solvents for drug analysis
are ones where the drug of interest is freely soluble

Green/brown

Grey/brown
and the solvent does not react with the drug or cat-
alyze drug breakdown. The solvent should also be
volatile and water free to allow for easy concentra-
tion of the sample. The sample to be analyzed
together with positive and negative (solvent only)
mecloqualone
Methaqualone

controls should all be spotted onto the same TLC

plate and developed in the usual manner.
After development the plate is viewed under UV

Blue
light and any spots present are marked. Other vis-
ualization techniques are commonly used and differ-
Orange/red

ent classes of drugs require different techniques

Mescaline

usually involving spraying the plates with various

reagents (Table 2). Rf values are determined and
compared with the controls run on the same plate
Red/purple/yellow

and may be compared with literature values (though

these should be interpreted with care). In the case of
Pink/yellow

Blue (slow)

trace samples, the samples are swabbed using a buc-

cal swab. The swab is first extracted (rinsed) with a
Benzo.

Yellow

solvent suitable for the analysis. The solvent is con-

centrated under nitrogen and analyzed for the pres-
ence of drugs. The swab is then rubbed over the
Slight green

Red/purple

surface of the item thought to contain the trace drug,

the head cut off and extracted with solvent, and the
Barb.

extract concentrated under nitrogen and analyzed.

The swab head and concentrated extract should be
Blue/violet

refrigerated and retained.

Despite being relatively cheap, rapid, and easy to
LSD

interpret, TLC has a number of disadvantages which

include:
Red/purple
Cannabis

* lack of resolution;
Violet
Table 1 Expected color reactions for the most common drug compounds

* lack of specificity of spray reagents giving similar

color reactions for some compounds of the same
Cocaine

class;
Orange

* edge effects on the TLC plate;

Blue

* variable Rf values; and

* relatively low sensitivity.
Brown/orange/green

Chromatographic Methods used as

Red/brown

Confirmatory Techniques
Amphet.

Green

Having identified the drug class and perhaps the drug

within that class, the next stage of the analytical
procedure is to confirm the identity of any drug
Purple/violet

present. Instrumental techniques are used to accom-

Red/brown
Opiates

plish this and these can be either chromatographic or

Drug

spectroscopic. Such techniques provide much stron-

ger evidence than noninstrumental techniques.
Chromatographic techniques identify drugs
Cobalt thiocyanate
Duquenois-Levine

through their physical and chemical properties of

Dille-Koppanyi
Zimmermann

the molecule as a whole such as structure and size,

Fast Blue B
Vitali-Morin
Mandelin

whereas spectroscopic tend to identify compounds

Marquis

Ehrlich
Simon

on the basis of certain parts present within the mol-

Test

ecule. Instrumental techniques are used not only in

 FORENSIC SCIENCES / Systematic Drug Identification 475

Table 2 Some suggested TLC systems and visualization reagents for different drug compounds

Drug TLC mobile phase Visualization reagent Result

LSD Chloroform/methanol (9:1 v/v) 1. UV at 254 nm Absorbs

2. UV at 360 nm Fluorescence
3. Ehrlich’s reagent Blue/purple
Opiates 1. Ethylacetate/methanol/ammonia 1. UV at 254 nm Absorbs
(85:10:5 v/v)
2. Chloroform/methanol (9:1 v/v) 2. Acidified iodoplatinate Blue/purple
3. Dragendorf solution Orange
Amphetamines Methanol/ammonia (100:1.5 v/v) 1. Ninhydrin reagent and heat at 1151C Violet/pink
2. Result of 1 þ acidified iodoplatinate Gray/violet/brown
3. 0.5 mol l
1 NaOH þ 0.5% Fast Brown/pink/red
black K
Barbiturates 1. Ethylacetate/methanol/25% ammonia 1. UV at 254 nm Absorbs
(85:10:5 v/v)
2. Chloroform/acetone (80:20 v/v) 2. Mercury(II) chloride–
diphenylcarbazone
Cocaine Methanol/25% ammonia (100:1.5 v/v) Acidified iodoplatinate Blue/pink
Mescaline/ Methanol/ammonia (100:1.5 v/v) 1. UV at 254 nm Absorbs
psilocin
2. Fluorescamine reagent Bright at 365 nm
3. Ninhydrin reagent and heat at 1201C Violet

confirming the identity of a drug, which may be
present but also in quantification (determining the
concentration) of the drug which may be present.
Liquid Chromatography of Drugs of Abuse
LC is a technique used commonly to identify and
quantify drugs of abuse. The technique has a number
of advantages and disadvantages specific to drug
analysis.
Advantages:
* it is nondestructive and samples can be recovered
if required;
* the analyte does not need to be volatile;
* the sample generally does not require pretreat-
ment such as chemical derivatization;
* the analysis can be automated; and
* quantification can be achieved without the neces-
sity of an internal standard.
Disadvantages:
*
the analyte needs to have properties which can be
detected in a liquid stream;
* in most cases a UV or diode array detector and so
the analyte needs to possess chromophores;
* the sample needs to be soluble in a wide range of
solvents;
* quantification can be slow; and
* large volume of solvents are used.
In general a mixture of reverse-phase and straight-
phase systems are used depending on the drug under
test. LC analysis is particularly used where quanti-
fication of the target drug is required as the system
can be run without the use of an internal standard.
Difficulties arise in complex mixtures of street drugs
where reaction by-products and impurities as well as
additives can cause complications within the chro-
matogram. As in any forensic examination, the in-
jection sequence for samples is of importance.
Calibration standards and samples should always
be interspersed by blank injections to ensure clean-
liness of the instrument.
Gas Chromatography
GC has advantages and disadvantages as a confirm-
atory technique when compared to LC. The GC sys-
tem has a greater resolving power than LC systems
and does not have the same problems associated with
mobile phase choice or require large volumes of
solvents. However, GC does require the compound
to be thermally stable, volatile, and exhibit good
chromatographic qualities. This often requires
derivatization of the compounds.
Chemical Derivatization of Some Drugs
Many drugs can be chromatographed using GC di-
rectly; however, a number of compounds may give
rise to problems such as thermal decomposition
(cannabinoids and cocaine alkaloids), reactions with-
in the instrument between compounds in the injected
mixture (morphine), adsorption, or coelution. In or-
der to improve chromatographic results some drug
compounds require derivatization where –OH,
476 FORENSIC SCIENCES / Systematic Drug Identification
–NH2, and –COOH groups are modified. The choice
of derivatization reagent depends upon the types of
functional groups present. Derivatization alters the
chemical structure of the compound to a certain
degree, which will in turn influence the results of any
subsequent mass spectra produced. There are some
common derivatization reagents in use:
1. N,O-bistrimethylsilylic acid (BSA) is commonly
used to derivatize cannabinoids, opiates and
cocaine analogs (ecognine, etc., cocaine shows
good chromatography).
2. N,O-bistrimethylsilytrifluoroacetamide (BSTFA)
is commonly used to derivatize LSD and psilocy-
bin.
3. Trifluroacetic anhydride (TFAA) and heptabutyrl
anhydride (HFBA) are commonly used to derivat-
ize amphetamines.
GC and LC provide a means of separating the
components of a complex mixture but neither tech-
nique can definitively identify any component. More
than one component may have the same retention
time for a given system or may have chromophores,
which are difficult to differentiate. For this reason it
is generally accepted that two (preferably three) in-
dependent techniques are used to identify the com-
pounds present. The generation of UV absorption
spectra using diode array detection (DAD) can help
accomplish this in LC analysis though many com-
pounds have similar UV spectra. In GC analysis, the
system can be coupled to a mass spectrometer and a
mass fragmentation pattern produced for each com-
pound, which can be used to identify the compound
together with the chromatographic data.
GC–FID is often used in drug analysis but increa-
singly routine is the use of GC–MS. In this case the
GC is interfaced with a mass spectrometer detector
(MSD). This can be either in place of a conventional
detection system such as an FID or in tandem with an
existing detector. When using an MSD the carrier gas
is generally helium, which has low molecular weight
(does not contribute to observed ion signal) and
which is oxygen and water free.
The information derived form GC–MS includes
retention time and the mass spectrum of each eluted
peak. Principal peaks are used in the identification of
unknown compounds where these are the peaks of
the most intense ions (usually 6–8 peaks). These can
be compared to reference data in an attempt to iden-
tify the compounds.
Selective ion monitoring If a drug sample being
analyzed is complex, it may be difficult to obtain
baseline resolution for the different components of
the mixture. Selective ion monitoring essentially
means setting the mass spectrometer to examine the
mass spectra produced for a particular ion peak or
set of peaks. The total ion chromatogram (TIC) can
then be scrutinized for the presence of these peaks.
This method can be problematic. The selected ions
only form a small part of the total ion count. If the
sample is weak then the selected ions may not be
visible against the background noise. Secondly ions
chosen must be characteristic of the compound in
question.
General comments Quantification can be carried
out using a GC–MS (or a GC–MS with FID detector)
either by using the internal standard technique or
using a deuterated homolog (compound of similar
structure) as the compound under test. Generally the
methyl group attached to a nitrogen moiety or func-
tional groups attached to the main structure are
those which are deuterated. The replacement of hy-
drogen atoms in this manner alters their mass from 1
to 2, thus causing different fragmentation patterns.
This removes the necessity for baseline resolution of
the target peak and its deuterated internal standard.
Quantification is determined using the ratio of the
peak heights (or areas) of the specific ions within the
mass spectrometer.
Spectroscopic Techniques
Fourier transform infrared spectroscopy FTIR is an
extremely useful technique for confirming the iden-
tity of pure compounds, but has limited value if used
for mixtures of compounds. The technique is based
upon the identification of functional groups within
molecules where such groups vibrate (either through
stretching or bending in various ways) when irradi-
ated with specific wavelengths of light. These vibra-
tions and their intensity (% transmission) are plotted
against the frequency of light (cm
1) to which the
sample is exposed to produce an FTIR spectrum.
Portions of the FTIR spectrum are unique to the
compound under test (this is called the fingerprint
region). Unfortunately, because the majority of seized
samples are mixtures of compounds, FTIR has lim-
ited practical use in the analysis of street samples of
drugs of abuse. However, it does have the advantages
of being nondestructive and not requiring derivat-
ization.
Ultraviolet–visible (UV–Vis) spectroscopy UV–Vis
spectroscopy, like FTIR, is a technique which is use-
ful in the identification of pure drug compounds.
Different compounds contain chromophores, which
will absorb specific wavelengths of UV or visible
 FORENSIC SCIENCES / Systematic Drug Identification
light. The technique obeys the Beer–Lambert law and
as such the absorption of spectra generated at given
wavelengths have the added advantage of being di-
rectly related to the concentration of the sample (this
is the basis of the diode array detector used in LC).
Normally UV and UV–Vis spectra are recorded at
high and low pH and the results of both for the sam-
ple under question compared with known standards.
UV–Vis is a cheap and easy technique, which al-
lows sample recovery and good discrimination
between pure compounds without the need for
derivatization. It has less application for street sam-
ples involving complex mixtures.
Which Instrumental Technique
to Use?
The choice of instrumental technique used in any
analysis depends upon the motive behind the analysis
and on the nature of the sample. Normal procedure
when dealing with bulk samples is to perform pre-
sumptive color tests to identify the drug class
followed by TLC to identify the specific member of
the drug class. When dealing with trace samples, it is
often the case that these nonconfirmatory tests are
circumvented and confirmatory tests only are em-
ployed. The choice of confirmatory test now depends
on the motive behind the analysis as indicated below.
The choice of technique will also depend upon the
nature of the analytes under investigation.
For example, large compounds such as the can-
nabinoids are not particularly thermally labile and
benefit for derivatization, however, this has implica-
tions in drug profiling as derivatization results in
altering the chemical structure. LC analysis of can-
nabinoids will effect their identification but difficul-
ties in resolution may hamper quantification and
profiling. In practice many laboratories now identify
cannabis on the basis of microscopy rather than in-
strumental techniques.
Samples thought to contain heroin require derivat-
ization to help resolve some of the components
though diamorphine itself chromatographs well
without derivatization. Introduction into the GC
system causes acetylation of morphine and mon-
oacetyl morphine thus inflating the concentration of
diamorphine above the actual value if GC is used as
the quantifying technique. Heroin samples are gene-
rally profiled using a GC/MS system.
Amphetamine compounds are generally easily
analyzed using either GC or LC and are profiled
using a GC/MS system. LC is useful for quantifica-
tion though a common diluent, caffeine has a signi-
ficantly different molar extinction coefficient than
477
the amphetamines causing a large peak on the chro-
matogram, which may cause coelution problems.
There are also coelution problems with some of the
structurally similar compounds.
Barbiturates and benzodiazepines are difficult to
analyze both by GC and LC. In GC analysis, barbit-
urates are derivatized on column by the addition of
0.2 mol l
1 trimethylanilium hydroxide in methanol
and concentrating the solution using N2. The sample
is injected into GC where and alkylation reaction
takes place. The sample retention is governed by the
length of carbon chain at the C-5 position and the
degree of polarity of the molecule. In general as the
carbon chain length increases, so does the lipophili-
city and consequently the retention time. Where
chain lengths are the same, the degree of polarity
becomes the deciding factor with more polar com-
pounds eluting earlier.
Data Interpretation
There are a number of mathematical techniques
commonly used with data obtained from instrumen-
tal techniques. These enable the verification of the
instrumental analysis with respect to reliability and
reproducibility of response, comparison of retention
time data, and the determination of the concentra-
tion of active component (to greater or lesser degrees
of accuracy and with slight variation depending on
the type of analytical technique used).
Relative Retention Time
The relative retention time (RRT) is often calculated
for peaks in chromatograms particularly when its
identity is unknown. This can be compared to other
sample data or data generated using standards or
from a database generated under the same chro-
matographic conditions. An internal standard is add-
ed to the test solution and the following equation is
used. RRT values can be calculated for both GC and
HPLC analysis:
Relative retention time
Retention time for analyte
¼ ½2
Retention time for internal standard
It is important to be able to measure the reliability
and reproducibility of analysis. This is usually con-
ducted on a daily or more often weakly basis by
analyzing standard samples. Both repeat injections of
the same standard (showing instrumental variation)
and different samples of the same standard (showing
extraction and sample variation) and apply an
appropriate statistical analysis to the results.
478 FORENSIC SCIENCES / Systematic Drug Identification

Relative Standard Deviation (Coefficient
of Variation)
This is a measure of the standard deviation expressed
as a percentage over a number as of analytical re-
sults. The relative standard deviation (RSD) value
(%) should ideally be as low as possible showing a
high analytical reproducibility.
s
RSD ¼ 100
x%
Quantification Techniques
Single Point Estimates
This method is one of the quickest that can be used,
however, an assumption is made that the instrumen-
tal response is proportional to the concentration of
the analyte under test and that the concentration of
the sample is within the linear dynamic range of the
detector. One standard solution is analyzed along
with the sample and eqn [4] is used to relate their
concentrations to each other. Equation [4] illustrates
the application for LC analysis; if GC analysis were
being carried out each solution would also include an
internal standard.
Peak area sample Concentration of sample
¼ ½4
Peak area standard Concentration of standard
Peak area sample
 Concentration of standard
Peak area standard
¼ Concentration of sample
Two-Point Estimates
In this case two standards are analyzed, one of a
concentration higher than is expected in the sample
and one of a concentration lower than expected.
Again the assumption is made that all measurements
are made within the linear range of the detector. The
relationships between the different standards and
their respective responses are solved as simultaneous
equations to determine an equation of the straight
line between the points. The concentration of the
sample is determined using this equation.
½3 Regression Analysis
This is by far the most accurate method for quan-
tifying samples. It does not rest on the assumption
that the concentrations determined are within the
linear range of the detector but rather verifies wheth-
er this is correct. The method also involves running
repetitive samples of the same standard and as such
will take into account variation of detector response.
However, the method does require analyzing many
samples (at least 2 and preferably 3 data points at
five different concentrations) and as such can be time
consuming.
Techniques in Drug Profiling
In the clandestine preparation of illicit drugs, specif-
ically amphetamine and its analogs a number of
common synthetic routes are utilized (Figure 1).
As a consequence of this route, specific impurities
can be identified in GC analytical traces obtained
from submitted samples and these can be used to
determine which manufacturing route was utilized as
well as in investigating links between illicit samples
through impurity profiling.
Organic Impurity Profiling
At the present time, chemical profiling methods
are mainly based on determining and quantifying

CH2 CH3 CH2 CH3

Formic acid
C + OHC − NH2 C
O N
H COH
P2P Formamide N- formylamphetamine
Stage 1− Formylation reaction.

CH2 CH2 CH3

CH3
C Acid C
N NH2
H COH
N- formylamphetamine Amphetamine

Stage 2 − Hydrolysis reaction.

Figure 1 Leuckart synthesis of amphetamine.

 FORENSIC SCIENCES / Systematic Drug Identification 479

organic impurities present in the seizure. In the case
of synthetic drugs, some of these impurities and in-
termediates may be route specific. Techniques such
as GC–FID, LC, and FTIR may be employed but
perhaps the most beneficial is GC–MS, whereby, the
identity of eluted components can be determined
from the corresponding mass spectrum. Ultimately,
determination of the organic impurities aims to iden-
tify common batch links among illicit seizures, while
quantification has ramifications in subsequent crim-
inal proceedings. Illegally synthesized drugs may
eventually be distributed in more than one country
and hence, the need for a universal method to es-
tablish links between seizures is apparent. Recent
work has focused on the development of computer-
based programs to improve the accumulation and
appropriate accessibility of drug intelligence data.
Early chemical profiling attempted to establish
batch links among illicit drug seizures by compa-
rative studies, based on both qualitative and
quantitative information, although success in ac-
hieving this has been varied. Such studies have been
applied to link heroin and cocaine samples to com-
mon origins, based on the concentration of the
organic impurities present. Isotopic analysis of im-
purities and intermediates has been employed in the
comparison of seized heroin, methamphetamine, and
ecstasy samples. X-ray diffraction and NMR spec-
troscopy (both 1H and 13C) have been used to
differentiate the origin of illicit ecstasy seizures
(Figure 2).
Inorganic Impurity Profiling
Since 1980, ICP-MS has emerged as a major and
powerful technique in the area of elemental analysis.
It offers extremely low detection limits which range
from sub parts per billion (ppb) to sub parts per tril-
lion. In addition, these detection limits are broadly
achieved for almost all the elements across the pe-
riodic table. Also, the simple nature of the mass
spectra of the elements makes this technique a quick
tool for automated qualitative, semiquantitative, and
quantitative elemental analysis. Metal content of
bulk drug substances may origin from different
sources:
* from the plant containing the drug which is then
extracted (Poppy, Cannabis, or coca leaf);
* from catalysts, reagents, and solvents used in the
extraction and/or syntheses;
* from exposure to air-borne particles;
* from container/closure systems, etc. used in syn-
thesis/extraction.
There exists, therefore, a potential for use of this
technique in relation to drug profiling. Moreover,
easy sample preparation (powders directly dissolved
in dilute nitric acid, with or without help of

Abundance 02020111.D\FID1A Abundance 0202409.D/FID1A

5 800 000
4e+07 5 600 000
3.8e+07 5 400 000
5 200 000
3.6e+07 5 000 000
3.4e+07 4 800 000
3.2e+07 4 600 000
4 400 000
3e+07 4 200 000
2.8e+07 4 000 000
2.6e+07 3 800 000
3 600 000
2.4e+07 3 400 000
2.2e+07 3 200 000
2e+07 3 000 000
2 800 000
1.8e+07 2 600 000
1.6e+07 2 400 000
1.4e+07 2 200 000
2 000 000
1.2e+07 1800 000
1e+07 1600 000
1400 000
8 000 000
1200 000
6 000 000 1000 000
4 000 000 800 000
600 000
2 000 000
400 000
200 000
Time 4.00 8.00 12.00 16.00 20.00 24.00 28.00 32.00 36.00 Time 4.00 8.00 12.00 16.00 20.00 24.00 28.00 32.00 36.00

Figure 2 GC–TIC of impurity profiles of two unrelated (street) amphetamine samples.

480 FORENSIC SCIENCES / Systematic Drug Identification
14.68
43.12
Similarity
71.56
100.00
92 92 101 101
A B A B
Figure 3 HCA of ecstasy tablets indicating linkages between samples.
microwave heating) and quick analysis time (about
2 min per sample) make this technique very attractive
for drug profiling.
Isotope Ratios
Stable isotope analysis is based on the fact that the
major elements of organic compounds exist in their
naturally occurring isotopic form. The corresponding
stable isotope ratios (18O/16O, 15N/14N, 13C/12C,
and 2H/1H) in natural products depend not only on
the biosynthetic pathway but also on the environ-
mental conditions such as humidity or temperature.
Consequently, it is possible to use such analyses to
determine the geographical origin of natural prod-
ucts. They are already routinely used by many food
control laboratories to detect, for instance, addition
of foreign sugar in pure fruit juices. For synthetic
substances, the isotope ratios are linked to the syn-
thetic conditions (reagents, time, catalysis, etc.) used
and to the purification processes, e.g., distillation and
it is possible to discriminate between batches of
substances. For instance, analgesic drugs such as
acetaminophen and aspirin, even manufactured
following precise protocols, are significantly hetero-
geneous in isotopic composition. One advantage of
stable isotope analysis is that isotope ratios are in-
trinsic parameters of the drug substance; therefore,
they are not modified by partial degradation or ad-
dition of foreign materials during the distribution
process.
Data Analysis in Drug Profiling
The power of chemical profiling has been further
enhanced through the application of chemomet-
ric procedures to the analytical data generated.
143 102 102 143 158 158
A B A B A B
Seizure
Amphetamine samples have been linked, according
to the impurities present, using the Quotient method
and a supervised learning method known as SIMCA
(soft independent modeling of class analogy), both of
which were applied to develop a harmonized method
for heroin profiling. Canonical variate analysis has
also been used to classify methylamphetamine hy-
drochloride according to synthetic route, again based
on the chemical impurities present and multivariate
methods of hierarchical clustering, principal compo-
nent analysis, and k-nearest neighbors were utilized
to establish batch links in illicit heroin samples
(Figure 3).
See also: Chemometrics and Statistics: Multivariate
Classification Techniques. Forensic Sciences: Drug
Screening in Sport; Illicit Drugs; Thin-Layer Chro-
matography. Gas Chromatography: Overview; Mass
Spectrometry; Forensic Applications. Liquid Chro-
matography: Clinical Applications. Microscopy Appli-
cations: Forensic. Spot Tests.
Further Reading
Cole MD (2003) The Analysis of Controlled Substances.
Chichester: Wiley.
Gough TA (1991) The Analysis of Drugs of Abuse. Chich-
ester: Wiley.
Laing R (2003) Hallucinogens, a Forensic Drug Hand-
book. New York: Academic press.
Moffat A, Osselton MD, and Widdop B (2003) Clarke’s
Analysis of Drugs and Poisons. London: The Pharma-
ceutical Press.
Tebbett I (1992) Gas Chromatography in Forensic Science.
Chichester: Ellis Horwood.
Yinon J (1995) Forensic Applications of Mass Spectro-
metry. Boca Raton, FL.