This book brings together information that is not usually available collectively and enables the reader to engage with current debate in the field of addiction science.
It will be relevant for Institutional Library collections and Academics and Postgraduates working in the fields of Analytical, Chromatography, Medicinal and Pharmaceutical chemistry; Drug metabolism; Addiction science; and Forensic Toxicology, Science and Medicine.
The following societies and websites may be approached to publicise the book:
LTG (London Toxicology Group), TIAFT (The International Association of Forensic Toxicologists), IATDMCT (International Association of Therapeutic Drug Monitoring and Clinical Toxicology), RCPath (Royal College of Psychiatrists), Institute of Pharmaceutical Sciences, Royal College GPs; Society for the study of Addiction; EUROPAD (European Opiate Addiction Treatment Association) annual conference- www. europad.org; ISAM-International Society Addiction Medicine; ECMDDA- European Monitoring centre for Drugs and Drug addiction-http://www.emcdda.europa.eu/.
Cross promotional opportunities: Issues in Toxicology Series, The Forensic Chemistry of Substance Misuse, The Misuse of Drugs Act
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About the Author
Dr Kim Wolff, a Reader in Addiction Science, is an expert in biomarkers of abused substances and was chair of the expert panel on drug driving published by the Department for Transport (March 2013). She is also a member of the Secretary of State for Transport ‘Honorary Medical Advisory Panel on Alcohol, Drugs and Substance Misuse & and Driving’ and has worked closely with the DVLA over a number of years advising on medical fitness-to-drive issues and the High Risk Drink driver offender scheme.
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Urinalysis: The Detection of Common Drugs in Urine
Michael David Osselton
1.1 Introduction and Historical Background
Mathieu Orfila, Robert Christison and Alfred Swaine Taylorwere amongst the first early toxicologists in the 18th and 19th centuries to record the use of urine as an aid to poison detection.
"One of the strongest proofs of poisoning in the living subject is the detection of poison by chemical analysis in the matters vomited or in the urine, if the poison be one of those which are eliminated from the kidneys. The evidence is, of course, more satisfactory when the substance is discovered in the matter vomited or in the urine because it will show that the poison has really been taken, and will at once account for the symptoms."
At the time of Orfila, Christison and Swaine Taylor, the mechanisms by which poisons exerted their action in the body were unknown, and tests for the presence of poisons in urine were largely associated with administering the urine of suspected poisoning victims to animals, yet urine in the diagnosis and detection of drugs and poisons was later recognised as a valuable matrix for toxicological investigation. Only after the introduction of gas chromatography (GC) into forensic laboratories in the 1960's were toxicologists able to move away from urine in order to explore the use of blood, where better interpretation can be made from analyses. Today, urine still occupies an important place in forensic, sports, clinical and workplace drug testing, thanks largely to developments in high-performance liquid chromatography linked to mass spectrometry (HPLC-MS).
1.2 Urinary Drug Excretion
Urine is produced in the kidneys and comprises water (>95%) together with the waste products of metabolism. The kidney plays a key function in maintaining the health of an individual by regulating the salt and water balance in the body and providing a route for the elimination of metabolic excretion products and toxic substances. Urine comprises a concentrated solution of filtered waste products that pass out of the body via the bladder. In the healthy individual, the urine is a clear, pale yellow fluid containing water (>95%), urea (~2%), creatinine (~0.1%; >2 mmol L-1 or >226 mg L-1) and small quantities of salts, together with water-soluble drugs and their metabolites. The normal glomerular filtration rate is approximately 120 mL minute-1. After reabsorption of salts, glucose and water in the proximal tubules of the kidney, the filtrate reaches the loop of Henle with the same osmotic pressure as plasma at a rate of approximately 20 mL minute-1. After reabsorption of water in the distil tubules, the final urine flow is less than 0.5 mL minute. The rate of urine production varies with age, such that a 1-week-old baby produces 50–800 mL 24 hours-1, a 3-year-old child produces 500–700 mL 24 hours-1, a 10-year-old produces 700–1400 mL 24 hours-1 and a healthy adult produces 800–2000 mL 24 hours-1.
Normal urine has a specific gravity of greater than 1.025. Specimens with a specific gravity of less than 1.001 may indicate dilution caused by excessive fluid consumption, diabetes insipidus, impaired renal function resulting from a low number of functioning glomeruli or, in workplace drug testing scenarios, an attempt by the donor to dilute their urine specimen to try to confound the testing process. Although specific gravity is infrequently measured in post-mortem and criminal toxicology, it is regularly measured in workplace drug testing laboratories as a test for deliberate specimen dilution. The pH of urine is naturally variable and may range between 4.5 and 8.0.
Depending on the pH of the urine and the state of ionisation and/or water solubility of any drugs or their metabolites present in the glomerular filtrate, drugs and their metabolites become trapped in the urine and are subsequently excreted from the body. Although drug concentrations in the glomerulus are similar to those in blood, the reabsorption of water back into the blood as the urine passes through the convoluted tubules and loop of Henle results in increased drug concentrations in urine, such that drug concentrations may be between 10 and 100 times more concentrated in urine than those in the circulating blood.
As a matrix that is largely aqueous and free from proteins and cells in normal healthy individuals (cf. blood), where drug concentrations are usually significantly higher than in blood and where drugs may accumulate prior to elimination from the body, urine provides an ideal sample for drug screening. As a consequence of this concentration effect, drugs and their metabolites may be detected in urine long after they become undetectable in blood; hence, urine analysis provides an extended window of detection for the forensic or clinical analyst. Approximate time intervals for the detection of drugs in urine following single dose consumption are shown in Table 1.1.
1.3 Urine Collection and Storage
Urinalysis generally involves two stages (i.e. screening and confirmatory analysis). Sample collection is guided by the type of analysis to be performed and may differ depending upon whether the sample is to be collected for clinical, workplace or forensic purposes. Forensic screening usually requires analysis for the presence of alcohol, whereas clinical compliance testing, emergency screening of patients admitted to casualty following adverse reactions to drug use and samples collected for workplace drug testing may require drug screening only. Samples requiring alcohol analysis should ideally be collected into glass containers with tight fitting tops containing sufficient sodium fluoride to produce a final fluoride concentration of between 1.5% and 2% w/v. Sodium fluoride acts as an efficient microbial growth inhibitor and subsequently prevents the formation of post-mortem alcohol from yeasts and the microbial degradation of drug metabolites.
In forensic cases, the sample should ideally be divided into two portions: one for alcohol analysis and a second for drug screening. In post-mortem cases, it is usually possible to collect at least 10–20 mL of urine, although in many instances the bladder may have emptied as a consequence of muscle relaxation following death and subsequent movement of the body. Specimens for clinical testing are usually collected into 25 mL sterile containers, and for workplace testing, a range of containers are commercially available, many of which incorporate lateral flow immunoassay strips for indicating whether commonly misused drugs are present in the specimen. Such containers may also incorporate a tamper-evident locking device in the lid, providing evidence of sample integrity between collection and receipt at the laboratory (Figure 1.1). Samples should be stored at 4 °C or alternatively frozen until analysis; however, for storage in the freezer, care must be taken to allow sufficient room in the top of the vial for expansion of the urine to avoid breakage of the container.
In the clinical scenario, urinalysis may be used for drug misuse screening or for monitoring patient compliance with prescribed medication, usually in the treatment and management of pain. Clinical compliance testing is undertaken to monitor illicit drug use in drug-dependent individuals and also to check that prescribed opioids are being taken in accordance with the physician's prescription and not syphoned off for illicit drug use. In general, clinical compliance testing incorporates analysis for a wider range of drugs than workplace testing, yet in common with workplace and forensic testing involves a two-step process (i.e. immunoassay screening followed by confirmatory analysis using GC-MS or HPLC-MS). Workplace testing is undertaken to identify individuals that use or misuse illicit drugs, particularly in safety-related occupations.
The extended window of detection offered by urinalysis coupled with generally higher concentrations of drugs than those measured in blood also makes urine an excellent screening matrix in cases where drug-facilitated crime (DFC) is suspected. In DFC cases, urine is usually plentiful and may be collected without the need for a nurse or medical practitioner to be present. This makes urine particularly valuable in cases where a subject may suspect that they have been drugged, since specimen collection can be undertaken without the requirement of medically trained personnel. In such cases, there may be a considerable time lapse between the suspected drug consumption and the subject reporting the incident; hence, the time between the ingestion of a drug and collection of specimens may be several hours or even days.
The extended window of detection for drugs in urine facilitates the detection of drugs and their metabolites long after they have fallen below the limits of detection in blood. In DFC cases, police and healthcare professionals are advised to collect an early urine specimen for analysis as soon as an alleged victim reports with a suspicion of drug-related assault. This should be followed up by the collection of a second urine specimen together with a specimen of blood when the victim is medically examined. The collection of an early urine specimen increases the chance of drug detection in cases where a drug possessing a short elimination half-life may have been consumed (e.g. γ-hydroxybutyrate). It is recommended that at least 25 mL of urine should be collected on both occasions and that 5–10 mL aliquots of the urine from each collection should be preserved with sodium fluoride to a final fluoride concentration of 1.5–2% w/v for alcohol analysis.
1.4 Interpretation of Analytical Results
The detection and quantification of a pharmacologically active drug in blood provides information that may be related to therapeutic, toxic and behavioural effects, whereas the detection of drugs in urine enables the analyst only to state that the donor of the sample had consumed a drug substance at some time prior to the provision of the specimen. Although urine provides an excellent matrix for drug screening, the interpretative value of measuring urine drug concentrations is limited, and most toxicologists use the sample for qualitative analysis only. After drugs enter the bladder, they are no longer able to exert an effect on the donor and, with few exceptions, little is known about the relative proportions of drugs in urine to those in blood, hence measured drug concentrations in urine are not easily or reliably interpretable.
The concentrations of drugs and their metabolites in urine do not reflect blood concentrations or relate to the effects of the drug on the donor. Urinalysis does not enable toxicologists to say when or how much drug was consumed. Analysis of a single urine sample offers only a 'snapshot' in time. In a DFC investigation, the analysis of an early evidence sample followed by a second sample collected a few hours later may provide useful information in relation to when a drug may have been consumed by studying the pattern of metabolites between the two specimens.
Table 1.2 summarises the drug testing panels and concentration cut-offs recommended by the United States Substance Abuse Mental Health Services Administration (SAMHSA), the European Workplace Drug Testing Guidelines and clinical patient compliance testing. The core SAMHSA panel is widely accepted internationally; however, some testing providers may agree to modified panels with their customers in order to reflect local or national trends in drug use that differ from those of the USA.
As a non-invasive specimen for collection, urine has been used for workplace/occupational testing since the 1980's following the introduction of testing by the US military. Unlike forensic testing, where extensive screening may be undertaken, workplace drug testing is usually limited to a restricted panel of drugs that include those that are most commonly misused (e.g. cannabis and cocaine) and that are likely to have potential for adversely affecting an employee's performance.
1.5 Approaches to Screening
The analytical approach to urine analysis varies with the desired outcome of the test. Most clinical, forensic and workplace testing procedures employ a two-stage analytical process (i.e. immunoassay followed by confirmatory analysis). Colour and smell can provide some valuable clues to drug/poison presence, particularly in clinical and post-mortem cases. Common drugs that may result in coloured urine are summarised in Table 1.3.
A range of simple colour tests can also be used to indicate the presence of compounds not commonly included in immunoassay screens such as chloral hydrate (Fujiwara test), oxidising agents (diphenylamine), cyanides (sodium picrate) and salicylates (Trinders). An extensive list of colour spot tests is beyond the scope of this chapter, but is provided in Clarke's analysis of drugs and poisons.
Immunoassays provide relatively inexpensive and rapid means of screening, offer the option of a high degree of sensitivity and are available for use either in a laboratory or in near-subject testing. For near-subject testing, a wide variety of lateral flow immunoassay devices is available. Near-subject screening uses lateral flow test strips that can be incorporated into portable hand-held devices that are suitable for screening in clinics, the workplace and by police at the roadside. 'Test cups' capable of offering screening and preservation of the sample for laboratory confirmation are also widely available (Figure 1.1).
The lateral flow immunoassay is based on the principal of a competitive immunoassay in which immobilised drugs are laid down in strips across a membrane over which antibodies labelled with a visualisation reagent such as fluorescent nanoparticles or colloidal gold are carried along the membrane by capillary action. A range of immunoassay test systems has been marketed by different manufacturers.
The principal of the assay involves mixing a sample of urine with labelled antibodies prior to travelling along the membrane and past the immobilised drug strips. If a drug is present in the urine, it binds with the labelled antibodies prior to the urine reaching the immobilised drug, thus preventing a reaction between the labelled antibody and the immobilised drug. If no drug is present in the urine, reaching the immobilised drug, thus preventing a reaction between the labeled antibody and the immobilised drug. If no drug is present in the urine, the labelled antibodies bind to the immobilised drugs on the strip and may be visualised using an appropriate technique, which in the case of gold-labelled antibodies is observed as a pink/purple band on the test strip (see Figure 1.2). If a drug is present in the urine specimen, the labelled antibodies are prevented from binding with the immobilised drug and no coloured line is observed.
Laboratory-based immunoassay screening commonly involves the use of microplates comprising a number of wells into which antibodies have been immobilised. Urine, or an alternative matrix such as blood or oral fluid, is added to each of the wells, together with a fixed quantity of enzyme-labelled drug. The enzyme-labelled drug and any drug present in the urine compete for the immobilised antibodies and, after allowing the samples to incubate and compete for the antibodies over a fixed incubation period, the matrix is removed from the wells and the wells are washed. A dye substrate is then added that reacts with any enzyme-bound drug present in the well. The intensity of the colour produced is measured and is proportional to the amount of enzyme bound to the immobilised antibodies in each well. The intensity of the colour reaction is inversely proportional to the drug concentration in the sample.
A major disadvantage of immunoassay screening, with the exception of the Randox biochip immunoassay screening system, is that the range of commercially available assays is limited to the detection of commonly misused drugs (e.g. amphetamines, benzodiazepines, barbiturates, cannabinoids, cocaine, lysergic acid diethylamide [LSD], opiates and methadone). Whilst this is satisfactory for the majority of workplace testing applications, the limited range of assays is unsuitable for forensic laboratories that are required to employ a range of broader screening procedures involving GC, LC and MS.
Excerpted from "Detection of Drug Misuse"
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Table of Contents
Urinalysis: The Detection of Common Drugs in Urine; Point-of-care/Collection Testing: Application to Drugs of Misuse Testing; Analytical Advances in Drug Detection: Human Sports Drug Testing; Analytical Overview of Drug Detection: Civil Aviation; Detection of Misused Drugs: Natural and Synthetic Cathinones; Detection of Misused Drugs: Psychoactive Piperazines; Dried Blood Spots for Testing Drugs of Misuse; Drug Testing in Exhaled Breath; DNA/RNA Aptamers for Illicit Drug Molecules; Latent Fingerprints for Drug Screening; Microneedle Patches for Caffeine Detection and Quantification; Detection of a Single Drug Exposure in Hair; Ethanol Analysis in Blood, Breath and Urine: Interpreting the Results; 'Ecstasy' Tablets: Batch Matching for Forensic Drug Intelligence Puropses in Malta; The Usefulness of Metabolites in the Interpretation of Drug Test Results; The Usefulness of Metabolic Ratios in the Interpretation of Steroid Misuse; Neurohypophyseal Hormones and Drugs of Misuse