Systematic Drug Identification☆

Niamh NicDaéid, University of Dundee, Dundee, United kingdom

© 2018 Elsevier Inc. All rights reserved.

Introduction 1
Preparation of the Analytical Space 1
Physical Examination 2
Sampling Protocols 2
Liquids 2
Powders and Tablets 2
Non Confirmatory Tests 3
Presumptive Tests—Identification of Drug Class 3
Thin Layer Chromatography—Identification of Drugs Within a Class 3
Confirmatory Tests 4
Chromatographic Methods 4
Gas chromatography (GC) 4
High performance liquid chromatography (HPLC) 4
Detection Systems for GC and HPLC 5
Spectroscopic Techniques 5
Fourier Transform Infra-Red Spectroscopy (FTIR) 5
Ultra Violet-Visible Spectroscopy (UV-VIS) 5
Nuclear Magnetic Resonance Spectroscopy (NMR) 5
Which Instrumental Technique to Use? 5
Data Interpretation 5
Quantification Techniques 6
Single point estimates 6
Two point estimates 6
Regression analysis 6
Conclusions 6
Further Reading 6

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 controlled substances follows a set procedure involving; the preparation of
the analytical space within the laboratory; a physical examination of the item including its packaging; the use of a sampling protocol
depending on the nature of the samples presented and the analysis required and, finally, the chemical analysis of the sample. There
are various analytical techniques commonly used in the forensic examination of suspected drug samples and these include
presumptive testing (color/spot tests), thin layer chromatography (TLC), high performance liquid chromatography (HPLC), gas
chromatography mass spectrometry (GCMS), Fourier transform infra-red spectroscopy (FTIR) and ultra-violet spectroscopy (UV-
VIS). In more recent years, the significant rise in new synthetic drug compounds such as synthetic cannabinoid and cathinone
derivatives have resulted in the more mainstream use of nuclear magnetic resonance (NMR) spectroscopy within forensic science
laboratories.
The choice of which technique to use very much depends upon the objectives of the analysis. Generally, this includes the
identification or confirmation of the presence of a controlled substance. It may also include the quantification of that substance
(i.e., determining the amount of the controlled substance present) and it may include the chemical profiling or characterization of
synthetic by-products and/or reaction impurities within the sample with the aim of identifying synthetic pathways.

Preparation of the Analytical Space

The most fundamental and first step of the examination of samples suspected of containing controlled substances is the initial
preparation of the space where the analysis is to take place. Benches should be washed down with detergent to ensure that they are

☆
Change History: June 2018. N NicDaéid updated the text, tables and references. This is an update of N.N. Daéid, Forensic Sciences—Systematic Drug
Identification, In Encyclopedia of Analytical Science (Second Edition), edited by Paul Worsfold, Alan Townshend and Colin Poole, Elsevier, Oxford, 2005,
Pages 471–480, ISBN 9780123693976.

Encyclopedia of Analytical Science, 3rd Edition https://doi.org/10.1016/B978-0-12-409547-2.14457-9 1

2 Systematic Drug Identification

completely clean. Audits of the cleaning program are normally included within a forensic laboratory’s standard operating processes
and are often a requirement of laboratory accreditation.
Sample packages should be opened individually for examination and the bench should be cleaned in between subsequent
packages being opened. There should be frequent changes of gloves and careful contemporaneous notes kept on all procedures
undertaken.

Physical Examination

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 reported and a decision taken as to whether the analysis of that item should be continued as a consequence.
The physical description of an item, as well as the nature of the analysis undertaken, will depend upon whether that item is
considered a bulk or trace sample. 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 (e.g., on tablets) or marks on the items (e.g., on
blocks of resin or packaging materials), these should be fully described and would normally be photographed. 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 morphology of tablets in terms of size, markings etc., the presence of
different types of trichomes (hairs) in plant material and so on.
A trace sample is considered to be one which is barely visible to the naked eye. Samples such as this may be present on the insides
of reaction vessels, scales, knives and other drug paraphernalia. In many cases, trace samples are swabbed from these items and are
analyzed using confirmatory techniques first. Such samples are very easily contaminated and significant care should be taken in
their analysis. Bulk samples require a sampling methodology 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 physical morphology (also called sampling by attributes). This means that if a seizure is found to contain, for
example, different types of materials, 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
cannabis resin are being sampled this should be away from the edges of the block as such edges can potentially be used to make
physical fits between the resin blocks. In cases where samples of powder are seized, it is necessary to ensure that the powder is
homogenous and there are a variety of methods commonly used to do this.
Two things generally dictate sampling protocols:

1. The legal obligation which may require that all items in a seizure are described and sampled.
2. The statistically appropriate number of samples to analyze such that a true representation of the variety of samples within a
seizure is reflected in the analyzed group.

The criteria for selecting the type of sampling protocol undertaken normally includes a balance between the loss of completeness
versus analysis time can cost. An international system of sampling which has been adopted and promoted by the United Nations
Office on Drugs and Crime (UNODC) is as follows:

Liquids
If all of one phase then a sample of the liquid is removed for testing. If there more than one phase present then a sample of each
phase should be removed for testing.

Powders and Tablets

1. Single package of material—The material should be removed from all packaging and weighed to a constant dry weight. The
sample is then homogenized and a sub sample removed for analysis.
2. More than one package—The material in each package should be examined for color differences. The contents of each package
should be weighed and each package tested using a color presumptive test or TLC. If it is assumed that all packages contain the
same material then;

(a) if there are less that 10 packages all should be tested;

(b) if there are between 10 and 100 packages, 10 should be chosen at random and tested;
(c) if there are more than 100 packages the square root of the total should be chosen at random and tested.
 Systematic Drug Identification 3

Non Confirmatory Tests

Presumptive Tests—Identification of Drug Class
The first test carried out on a sample are presumptive 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 solution or on adsorbent substrates. The most common type of
presumptive test involve the addition of various reagents to the sample to produce a color. Other tests involve the use of microscopy
to identify morphological features of plant materials such as trichomes in suspected cannabis samples, or microcrystalline tests
where the formation of specific crystals is indicative of the class of drug present. In all cases, appropriate positive and negative
control samples are also used. Table 1 lists some of the results obtained with different spot test reagents for common drugs of abuse.
Most presumptive tests are carried out on porcelain tiles. There are a number of clear advantages 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 unsuitable 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 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 thin layer
chromatography (TLC), a technique which has the advantages of being both rapid and cheap. The separation process depends on
the relative strength of interaction of the sample components 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 which is suitable for the analysis and suspected
compound.
Suitable solvents for drug analysis are ones where the drug of interest is freely soluble and the solvent doesn’t react with the drug
or catalyze drug breakdown. The solvent should also be volatile and water free to allow for easy concentration of the sample. The
sample to be analyzed together with positive (a standard of the target drug) and negative (solvent only) controls should all be
spotted onto the same TLC plate and developed in the usual manner.
After development the TLC plate is viewed under UV light and any spots present marked. Other visualization techniques are
commonly used and different classes of drugs require different techniques usually involving spraying the plates with various
reagents. Retardation factor (Rf) values are determined and compared with the positive controls run on the same plate and may be
compared with literature values (though these should be interpreted with care). In the case of trace samples, the samples are
swabbed and the swap extracted with a solvent suitable for the analysis. Some typical TLC systems are presented in Table 2.
Despite being relatively cheap, rapid and easy to interpret, TLC has a number of disadvantages which include a lack of resolution
and relatively low sensitivity, a lack of specificity of spray reagents resulting in similar color reactions for some compounds of the
same drug class. Edge effects can also occur on the TLC plate and the same sample can produce variable Rf values across more than
one TLC plate.

Table 1 Expected color reactions for the most common drug compounds

Drug

Methaqualone Psilocin
Test Opiates Amphet. Cocaine Cannabis LSD Barb. Benzo. Mescaline Mecloqualone Psilocybn

Marquis Purple/ Brown/orange/ Orange/ Green/

violet green red brown
Mandelin Red/ Green Orange Slight Pink/yellow
brown green
Duquenois- Violet
Levine
Dille- Red/
Koppanyi purple
Zimmerman Red/purple/
yellow
Vitali-Morin Yellow
Cobalt Blue Blue (slow) Blue
Thiocynate
Ehrlich Blue/ Gray/
violet brown
Simon Red/brown
Fast Blue B Red/
purple
4 Systematic Drug Identification

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 @ 254 nm Absorbs

2. UV @ 360 nm Fluorescence
3. Ehrlich’s reagent Blue/purple
Opiates 1. Ethylacetate/methanol/ammonia (85:10:5 v/v) 1. UV @ 254 nm Absorbs
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 & heat @ 115
C Violet/pink
2. Result of 1 þ acidified iodoplatinate Gray/violet/brown
3. 0.5 M NaOH þ 0.5% Fast black K Brown/pink/red
Barbiturates 1. Ethylacetate/methanol/25% ammonia (85:10:5 v/v) 1. UV @ 254 nm Absorbs
2. Chloroform/acetone (80:20 v/v) 2. Mercuric chloride—diphenylcarbazone
Cocaine Methanol/25% ammonia (100:1.5 v/v) Acidified iodoplatinate Blue/pink
Mescaline/psilocin Methanol/ammonia (100:1.5 v/v) 1. UV @ 254 nm Absorbs
2. Fluorescamine reagent Bright at 365 nm
3. Ninhydrin reagent & heat @ 120
C Violet

Confirmatory Tests
Chromatographic Methods
Having tentatively identified the drug class, normally the next stage of the analytical procedure is to confirm the identity of any drug
present. Instrumental techniques are used to accomplish this and these can be either chromatographic or spectroscopic. Such
techniques provide much stronger evidence than non-instrumental techniques and are generally required in order to provide
appropriately reliable evidence for the purposes of criminal proceedings in Court.
Chromatographic techniques identify drugs through the physical and chemical properties of the analytes as a whole, whereas
spectroscopic techniques tend to identify compounds on the basis of functional groups and other bonding within the structure of
the molecule. Instrumental techniques are used not only in confirming the identity of a drug which may be present but also in
quantification (determining the amount) of the drug which may be present.

Gas chromatography (GC)

GC systems rely on the target analytes being capable of being volatilized without decomposition and analyzed in the gas phase.
Separation generally occurs on the basis of molecular weight and polarity. Many drugs can be chromatographed using GC
directly, however a number of compounds may give rise to problems such as thermal decomposition (cannabinoids and cocaine
alkaloids), reactions within the instrument between compounds in the injected mixture (morphine), adsorption or co-elution of
compounds within a drug mixture. In order to improve chromatographic results, some drug compounds require derivatisation
where –OH, -NH2 and –COOH groups are chemically modified. The choice of derivatisation reagent depends upon the types of
functional groups present. Derivatisation alters the chemical structure of the compound which will in turn influence the results of
any subsequent mass spectra produced. There are a variety of common derivatisation reagents in use which include for example;

• N,O-Bistrimethylsilylicacid (BSA) is commonly used to derivatise cannabinoids, opiates and cocaine analogues (ecognine etc.,
cocaine shows good chromatography)
• N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) is commonly used to derivatise LSD and psilocybin.
• Trifluoroacetic anhydride (TFAA) and heptaflurobutyrl anhydride (HFBA) are commonly used to derivatise amphetamines.

High performance liquid chromatography (HPLC)

HPLC can be used as a non-destructive technique where samples can be recovered if required. The sample generally does not require
pre-treatment such as chemical derivatisation and the analysis can be automated rendering the process more efficient. Samples must
possess functional groups (chromophores) and properties which can be detected in a liquid stream, and the samples must be
soluble in a variety of solvents which can be used as mobile phases for the HPLC system. Quantification can be a relatively slow
process and the instrument can require large volumes of solvents.
In general, a mixture of reverse phase, straight phase and sometimes chiral chromatographic systems are used in HPLC
depending on the drug under test. HPLC analysis may specifically be useful where large molecules are targeted as these can be
challenging to analyze using GC. Difficulties can also arise in complex mixtures of street drugs which can be challenging to separate
from each other. In both GC and HPLC, calibration standards and samples are normally interspersed by blank injections of solvent
or mobile phase to ensure cleanliness of the instrument.
 Systematic Drug Identification 5

Detection Systems for GC and HPLC

While gas chromatography and high performance liquid chromatography provide a means of separating the components of drug
mixtures, neither technique can definitively identify the molecules within a sample mixture and 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 independent techniques should ideally be used to identify the compounds present. The generation of
UV absorption spectra using diode array detection (DAD) can help accomplish this in HPLC analysis though many compounds
have similar UV spectra. In GC analysis the system is usually coupled to a mass spectrometer (GCMS) and a mass fragmentation
pattern produced for each compound which can be used to identify the compound together with the chromatographic data. The
fragmentation pattern is derived from bombarding the target molecules with electrons as they emerge from the chromatographic
system, causing them to break apart. Such fragmentation occurs systematically and can be associated with a particular molecular
structure. The information derived from GCMS includes retention time and the mass spectra of each eluted peak. These can be
compared to reference data in for example, a library, in an attempt to identify the sample compounds.

Spectroscopic Techniques
Fourier Transform Infra-Red Spectroscopy (FTIR)
FTIR is an extremely useful technique for confirming the identity 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 irradiated with specific wavelengths of light. These vibrations and their
% transmission intensity are plotted against the wavelength of light (cm1) to which the sample is exposed to produce an FTIR
spectrum. Portions of the FTIR spectrum are unique to the compound under test (called the fingerprint region). Unfortunately,
because the majority of seized samples are mixtures of compounds, FTIR has limited practical use in the analysis of street samples of
drugs of abuse. However it does have the advantages of being non-destructive and does not require sample derivatisation.

Ultra Violet-Visible Spectroscopy (UV-VIS)

UV-VIS spectroscopy, like FTIR, is a technique which is useful in the identification of pure drug compounds. Many molecules
contain chromophores which will absorb specific wavelengths of ultra violet or visible light. Using the Beer Lambert law, the
absorption of spectra generated from these samples at given wavelengths can be related directly to the concentration of the sample.
Normally UV and UV-VIS spectra are recorded at high and low pH and the results of both for the sample under question compared
with known standards. UV-VIS is a cheap and easy technique which allows sample recovery and good discrimination between pure
compounds without the need for derivitisation. It has less application for street samples involving complex mixtures.

Nuclear Magnetic Resonance Spectroscopy (NMR)

NMR involves utilizing the physical properties of atoms to provide information about the molecular structure within which the
atoms reside. The basis of NMR is that, given nuclei within atoms are electrically charged and have a spin, then when they are placed
within an applied magnetic field, a measurable energy transfer can occur which can be used to generate an NMR spectrum. The most
common types of NMRs are focused on hydrogen atoms or carbon atoms and essentially provide a means for elucidating the
specific structure of the molecules based on the determination of the position of these atoms within the molecule.

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 presumptive color tests to identify the drug class followed
by TLC to narrow down the identity of the specific drug. When dealing with trace samples it is often the case that non confirmatory
tests are circumvented and confirmatory tests only are employed. The choice of technique will depend upon the nature of the
analytes under investigation and the strengths and limitations of the different techniques need to be carefully assessed.

Data Interpretation
There are a number of mathematical techniques commonly used with data obtained from instrumental analysis. These enable the
scientific validation of the analysis to be undertaken and the accuracy and precision of the technique as well as the measurement
uncertainty associated with the analysis of individual analytes to be determined. Analytical validation is a requirement of laboratory
accreditation.
It is important to be able to measure the reproducibility of analysis and this is usually conducted on a daily or more often weekly,
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) are undertaken as part of a normal laboratory quality
6 Systematic Drug Identification

control system which should be an essential component of forensic scientific procedure and is a requirement for external laboratory
accreditation.

Quantification Techniques
Single point estimates
This method is one of the quickest which can be used, however an assumption is made that the instrumental 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 Eq. (1) is rearranged and used to relate their
concentrations to each other.

Peak area sample Concentration of sample

¼ (1)
Peak area standard Concentration of standard

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 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 the resultant equation.

Regression analysis
This is by far the most accurate method for quantifying samples. It does not rest on the assumption that the concentrations
determined are within the linear range of the detector but rather verifies whether 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 two and preferably three data points at five different concentrations) and as such can be
time consuming.

Conclusions

The analysis of controlled substances is well researched and undertaken using robust and generally validated analytical chemical
methodologies. Most forensic laboratories which carry out such analysis are accredited under international standards to do so. With
the continued emergence of novel chemical compounds, however, forensic drug chemists face continued challenges to remain in
touch with new emergent compounds and to have their analysis facilitated with the availability of appropriate drug standards.

Further Reading

Laing, R. Hallucinogens, A Forensic Drug Handbook. Academic Press: USA, 2003.

Moffat, A.; Osselton, M. D.; Widdop, B. Clarke’s Analysis of Drugs and Poisons. Pharmaceutical Press: UK, 2003.
UNODC, World Drug Report. https://www.unodc.org/wdr2017/index.html; 2017.
Drummer, O. H.; Gerostamoulos, D. Forensic Drug Analysis. https://www.futuremedicine.com/doi/book/10.4155/9781909453371.
Moffat, A. C.; Osselton, M. D.; Widdop, B.; Watts, J. Clarke’s Analysis of Drugs and Poisons, 4th ed.; Pharmaceutical Press: London, 2011.