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Health and Life Sciences Faculty
Course Title: Biological and Forensic Science
Module code:
211 BMS
Module Title:
Principles of Forensic Science
The Analysis of Drugs of Abuse
Ciobanu Maria Alice
SID: 3395606
Word count:
1653
Principles of Drugs of Abuse Screening
Introduction
The mankind is exposed to numerous chemicals every day, whether we are aware of
it or not, and many of these are potentially harmful to us. In these circumstances, the
toxicology field progressed rapidly and diversified greatly, as it characterizes the
science of poisons with a focus on the understanding and study of the adverse
effects of chemicals or physical agents that may produce in living organisms under
specific conditions of exposure. (Hollinger; 1996) As part of this amazing science
domain, the term of ‘’forensic toxicology’’ was introduced to describe any application
of the science and study of poisons to the elucidation of questions that occur in
judicial proceedings. The field consists of three major subfields: post-mortem
forensic toxicology, human performance toxicology and forensic drug testing.
(Flanagan; 2007)
Although not exactly a common topic for social conversations, drug abuse has been
part of our society for centuries and remains a significant public health issue which
directly and indirectly touches the lives of many people. Major advances have been
employed in the understanding of neurobiology of addiction and the pharmacology of
abused drugs and still few tools effectively address drug abuse and addiction.
(Duffus; 1996) The term ‘’drug of abuse’’ is defined as any substance that is used
for a different purpose than the one intended, in order for some desirable effect to be
obtained. For example, the abuse of volatile solvents in what is known as ‘’gluesniffing’’. Also, it could describe any substance of which possession or supply is
restricted by law due to its potentially harmful effect on the user. (Flanagan; 2007)
However, a remarkable range of increasingly sophisticated technologies had been
employed in the fight against abused drugs over the past few decades. (Clarke et all;
2004) The competencies and dimensions for drug screening and toxicological
analysis have been greatly facilitated by the development of lateral flow
immunoassays which simplified the testing procedures and made test results
available in a timely fashion. Ligand-binding assays are commonly used for
screening. Techniques such as chromatography or electrophoresis are used as
separation
techniques.
Their
coupling
with
powerful
detectors
like
mass
spectrometry made them effective in confirmatory testing of preliminary positive
results. By these means, the pursuit of detecting, identifying and quantifying the
presence of abused drugs in a biological specimen with proper accuracy and
precision was successfully achieved. (Davis; 1987)
Drugs of Abuse Screening Technologies
Presumptive Test
Immunoassays are scientific tests that use the theoretical concept of antibodyantigen interactions to identify and measure amounts of a chemical substance in
biological specimens. (Levine; 2003) Many different immunoassays are available,
the main dissimilarity being the type of labelled compound and method of detection.
The most common type of assay used by toxicologists is enzyme-multipliedimmunoassay technique (EMIT).
Fig. 1 Schematic of EMIT (Drugs of Abuse, Coventry; 2013)
EMIT is a homogeneous assay which uses an enzyme-linked antigen. The basic
assay theory and typical standard plot are depicted in Fig.1. The enzyme G6P-DH
represents the label attached to the drug that oxidizes the substrate G6P to GL6P
and also reduces the cofactor NAD+ to NADH. Enzyme activity is determined by
spectrophotometrically measuring the produced NADH, while the absorbance is
measured at a wavelength of 340 nm. When the attached drug bounds to the
antibody, the enzymatic activity of G6P-DH decreases, therefore the process of
adding analyte reduces the antibody availability in terms of binding to the G6P-DH
labelled drugs and increases the rate of NADH production. Concentration of the drug
in the biological fluid analysed is directly related to the change in absorbance.
(James; 2000)
The advantage of EMIT is that it represents a non-radioactive assay together with no
requirement for separation of bounded and unbounded drugs. These homogeneous
assays are easy to automate since the reagents can be mixed, incubated and have
light measurements when made in the original reaction container. Once samples are
introduced, a typical ETS analyser can be programmed to perform a profile of tests
on each sample. This has the benefit of lowering costs by reducing the reagent
volume used. Automation also improves intra-assay variability and analyst error
liability. (Stine; 2006) Moreover, enzyme-related assays discriminate between
concentrations over a large scale. Change in absorbance of the solution is measured
as a function of time, hence the absorbance from interfering substances does not
usually change with time and their contribution is minimized.
Conversely, drug abuse immunoassays are often criticised for lacking selectivity.
Positive results with these systems carry a degree of uncertainty as interference can
result from compounds that cross-react with the antibody but also from substances in
the matrix that interrupt the enzyme process. For example, antibodies produced for
detecting amphetamine hapten may recognise other phenethylamines such as
ephedrine, an ingredient for common flu medicines. However, group-selective crossreactivity is not always a disadvantage. Amphetamine- reactive antibodies which
detect other abused stimulants are very useful, provided that a confirmatory test as
GC-MS is used subsequently to identify the drug present. (James; 2000)
Confirmatory Test
All specimens that tested positive on the presumptive test are subject to a rather
recent and innovative confirmatory technique called Gas Chromatography/ Mass
Spectrometry. Combined GC-MS is recognised as the reference technique for
identifying drugs and their metabolites in body fluids and it became the gold standard
method in order to avoid reporting false-positive results. (Stine; 2006)
Fig. 2 Overview of Gas Chromatography mechanism (Drugs of Abuse, Coventry;
2013)
Gas chromatographs can be fitted with a wide variety of detectors. The stationary
phase is a liquid and the carrier gas is generally an inert gas such as helium or
nitrogen. The inlet/ column system uses a dual type of columns: packed columns
and capillary columns. The injection port is usually heated independently. In order to
be analysed, various samples are directed into a port, where they are vaporized and
passed into the column (this represents the mobile phase). At this point of the
process, ions are being generated to produce an electronic signal that can be
recorded in order to create a gas chromatogram. (Jennings; 1978) Typically, a GC
shows a series of peaks that correspond to the components of the sample. Analysis
of defined compounds is obtained by comparing the retention time of a suspect
sample with that of a reference standard. (Rang; 2007)
Although gas chromatography allows the identification of substances based on
evaluation of Rf or Rt with standards, definitive recognition requires for the gas
chromatography to combine with mass spectrometry. Ideally, GC will provide a pure
compound which will facilitate the spectral analysis. (Plant; 2003)
When the components of the sample exit the GC column they enter the mass
spectral analysis stage. This is accomplished by measuring an analyte that has been
converted into a gas phase ion. Within the vacuum chamber, a beam of electrons will
continuously hit the analyte with the purpose of creating positive ions and breaking it
into ionic fragments. These fragments are easily affected by the electromagnetic field
through which are passed. This process allows the ions to be isolated with
remarkable specificity according with their mass/charge ratio. Each fragment is
specific for a given substance and the origin of the unknown component of a mixed
sample can be determined from the obtained spectrum plotting the abundance and
mass/charge ratio. (Jennings; 1978)
Direct combination of these two techniques described above constitutes an analytical
system of unparalleled capability, its greatest advantage being the exceptional power
of discrimination between closely similar structures such as the isomers (McMaster;
1998) GC-MS is extremely sensitive in detection and quantitative estimation, with an
impressive
separation potential of a mixed substance in a
relatively short
time.(Jennings; 1978)
However, there are also some disadvantages associated with GC/MS process. The
most obvious issue is related to the coupling of the two types of instruments because
of the difference in the operating pressures (GS effluent is at about 1 atm, whereas
MS is at or below 10-5 Torr), causing loss of sample or degradation of the
chromatographic separation. Choice of the stationary phase and operating
temperature is another limitation as they are controlled by the stability of the
stationary phase. (Jennings; 1978) If column bleed occurs, the ‘’background’’ ions
produced may hinder interpretation of spectra and limit the sensitivity in trace
analysis. In addition, the procedure can encounter difficulties of efficient trapping,
particularly in terms of minor components. Another disadvantage is represented by
the cost of the GS/MS equipment which is highly expensive and it requires careful
usage in order to minimize the practical issues arising from both constituent
techniques. Heat instability is another drawback of GC-MS when it comes to detect
tropane alkaloids such as atropine; therefore the compound first needs to be
converted to more stable derivatives. (McMaster; 1998)
On site testing- Cozart Rapiscan
A number of on-site immunochromatographic screening test systems for drugs have
been developed over the years. (Plant; 2003) The first on-site test used was the
electronic reading device known as the Cozart Rapiscan oral fluid testing system
which uses a lateral transfer immunoassay with colloidal gold antibody label. The
first step in the procedure is to collect the oral fluid in a device that will indicate the
sample adequacy and will retain it for further testing if required. The saliva
specimens are mixed with buffer and introduced in a fresh disposable cassette which
will be inserted into the hand-held instrument for incubation, reading and reporting.
The gold-labelled anti-drug antibodies contained within the cassette are rehydrated
by the saliva and run fluid. By capillary action, the mixture travels across an array of
immobilised drug sites. (Lucy; 2005) The lack of colour development at a certain
immobilised drug position suggests the presence of an illicit substance. The result
obtained is monitored by a portable reader and revealed on a display screen and
kept for further reference. If the immunoassay screening test for drugs in saliva is
positive, the sample will be send to a designed laboratory for confirmatory
chromatographic tests. (Clarke et all; 2004)
The market for on-site immunochromatographic assays is enormous and growing. In
terms of advantages, this screening device provided a convenient and inexpensive
tool for identification of target substances in biological specimens. Cozart Rapiscan
specifically combines easy and rapid sampling with fast analysis, requiring very little
cooperation of the person under evaluation, fact that makes it suitable for random
testing, for example at workplaces. (Clarke et all; 2004) Also, the results are digitally
displayed eliminating the user error and subjectivity and can be printed out on an
optional battery powered printer in order to keep a permanent record. Another
advantage is that it has an Internet interface module via which it up-loads constantly
new drug combinations, increasing its efficacy. Moreover, saliva represents a very
good specimen in drug detection as it reveals the unbound, non-ionised parent drug
or its lipophilic metabolites that circulate in the blood at the time of evaluation.
(Clarke et all; 2004) These are the forms of the drug that cross the blood-barrier and
affect performance and behaviour, therefore it is extremely useful for detecting
patient compliance with medication, driving under the influence of drugs, fitness for
duty, impairment for performance etc. It also offers the benefit of no adulteration or
substitution as it is performed under observation. (Clarke et all; 2004)
However, use of saliva concentrations has not yet reached the accuracy of blood
measurements. The device might display a false positive due to its incapacity of
distinguishing between legal and illegal drugs. Finally, without knowing the
instantaneous saliva pH, saliva drug concentration cannot be deduced to give blood
drug concentrations, which might describe a disadvantage. (Plant; 2003)
List of references
1 Coventry University (2013); Lecture for 211 BMS Principles of Forensic
Science; Drugs of Abuse Screening
2 Clarke, E. C. G. A. C Moffat; M Osselton; Brian Widdop, (2004); Clarke's
analysis of drugs and poisons : in pharmaceuticals, body fluids and postmortem material; 3rd edition; London : Pharmaceutical Press
3 Davis; R. (1987); Mass spectrometry ; Chichester: John Wiley & Sons
4 Duffus; J.H., Worth; H.G. (1996); Fundamental toxicology for chemists; 1st
edition ; Cambridge: Royal Society of chemistry
5 Flanagan; R.J., (2007); Fundamentals of analytical toxicology ; Chichester:
John Wiley & Sons
6 Hollinger; M.A., (1996); Toxicology: Principles and Applications ; 1st edition ;
London: CRC Press
7 James; R..C.; Roberts; S. M.; Williams, P.L. ( 2000); The Principles of
toxicology: environmental and industrial applications; 2nd edition ; Chichester:
Wiley
8 Jennings; W. (1978); Analytical gas chromatography; 2nd edition; London:
Academic Press
9 Levine; B. (2003); Principles of Forensic toxicology; 2nd edition ; Washington,
DC: AACC Press
10 Lucy; D. (2005); Introduction to statistics for forensic scientists; Chichester:
Wiley
11 McMaster; M.C. (1998); GC/MS: a practical user`s guide; 1st edition ;
Chichester: Wiley-VCH
12 Plant; N. (2003); Molecular toxicology; 1st edition ; Abingdon: BIOS
Scientific
13 Rang; H.P. (2007); Pharmacology ; 6th edition; Edinburgh: Churchill
Livingstone
14 Stine; K.E. ; Brown, T.M. (2006); Principles of toxicology ; 2nd edition;
London: CRC Press