The Analysis of Drugs of Abuse

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1 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 Word count: SID:

2 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)

3 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)

4 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)

5 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.

6 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

7 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

8 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

9 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)

10 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; 3 rd 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; 1 st 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 ; 1 st edition ; London: CRC Press 7 James; R..C.; Roberts; S. M.; Williams, P.L. ( 2000); The Principles of toxicology: environmental and industrial applications; 2 nd edition ; Chichester: Wiley 8 Jennings; W. (1978); Analytical gas chromatography; 2 nd edition; London: Academic Press 9 Levine; B. (2003); Principles of Forensic toxicology; 2 nd 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; 1 st edition ; Chichester: Wiley-VCH 12 Plant; N. (2003); Molecular toxicology; 1 st edition ; Abingdon: BIOS Scientific 13 Rang; H.P. (2007); Pharmacology ; 6 th edition; Edinburgh: Churchill Livingstone 14 Stine; K.E. ; Brown, T.M. (2006); Principles of toxicology ; 2 nd edition; London: CRC Press

Principles of Gas- Chromatography (GC)

Principles of Gas- Chromatography (GC) Mohammed N. Sabir January 2017 10-Jan-17 1 GC is a chromatographic technique utilizes gas as the mobile phase which is usually an inert gas (Hydrogen, Helium, Nitrogen