American Association for Clinical Chemistry
Better health through laboratory medicine
January 2008 Clinical Laboratory News: Drugs-of-Abuse Testing

January 2008: Volume 34, Number 1

Drugs-of-Abuse Testing
Understanding the Limitations
By Lynn Cheryk, PhD, and Loralie Langman, PhD




The Substance Abuse and Mental Health Services Administration (SAMHSA) estimates that 20.4 million Americans age 12 or older have used an illicit drug within the last month. According to the agency’s recent survey data, marijuana remains the most commonly used illicit drug, followed by prescription-type psychotherapeutic drugs, cocaine, and hallucinogens (1). Given the magnitude of drug abuse today, the need for laboratories to assist physicians by providing even the most basic identification of illicit drugs is great. The challenge to laboratorians, however, is to ensure that the physicians ordering the tests understand the information that labs can provide, as well as the limitations of the tests.

This article will focus on the currently available drugs-of-abuse (DOA) tests, the classes of drugs that are commonly analyzed, and the type of information obtained from these assays.

The Evolution of DOA Testing

The National Institute on Drug Abuse (NIDA), a component of the National Institutes of Health, was the first agency to issue mandatory procedural guidelines for workplace drug testing (2). NIDA designated urine as the specimen of choice for these programs because collection of urine is noninvasive and has fewer interfering proteins than other fluids or tissues (3). In 1988, the National Laboratory Certification Program (NLCP) was formed, spawning many of the current concepts, requirements, and methodologies for urine drugs of abuse (DAU) testing (2). In general, DAU testing involves rapid screening tests that detect broad classes of drugs to identify specimens as “presumptive positives.” The screening tests are followed by a more definitive and specific confirmation testing procedure.

DAU Screening and Confirmation Assays

Screening assays are generally rapid immunoassays based upon an antibody–antigen reaction involving the drug in question, a competing drug, and some type of detection system, such as the enzyme-multiplied immunoassay (Emit), fluorescence polarization immunoassay (FPIA), and the kinetic interaction of microparticles in a solution (KIMS) assay.

In the Emit assay, drug in the specimen competes with drug labeled with glucose-6-phosphate dehydrogenase (G6PDH) for antibody binding sites. Binding of the enzyme-labeled drug to the antibody results in decreased enzyme activity. As the concentration of drug in the specimen increases, the labeled drug is released from the antibody, increasing enzyme activity. Active G6PDH converts nicotinamide adenine dinucleotide (NAD+) to its reduced form, NADH, resulting in a change in absorbance at 340 nm (3). Any endogenous G6PDH present in the specimen does not react with the reagent as the coenzyme NAD functions only with the bacterial (Leuconostoc mesenteroides) enzyme used in the assay (4).

In the FPIA assay, fluorescein-labeled drug competes with drug in the specimen for antibody binding sites. Polarized light produces a polarized fluorescent emission from the fluorescein-labeled molecule. The amount of emitted fluorescence is related to the speed of rotation of the molecule. Rotation of the unbound fluorescein-labeled molecule through space occurs more rapidly than when the molecule is complexed to antibody. As the amount of drug in the specimen increases, the amount of bound to the labeled molecule decreases, and the amount of polarized light emitted decreases proportionately (5).

The KIMS assay uses changes in light transmission to measure the aggregation of microparticles in a solution. The microparticles have the drug of interest linked to their surfaces. In the absence of drug, antibody binds the drug-labeled microparticles, forming aggregates and increasing light transmission through the sample. When drug is present in the sample, it competes with the drug-labeled microparticles for antibody. Antibody binds to sample drug and is no longer available to promote particle aggregation, which decreases light transmission through the sample in proportion to the concentration of drug (3, 6).

The concentration of drug that distinguishes between positive and negative results in screening assays is referred to as the assay cutoff and is set at a concentration higher than the assay’s limit of detection. The screening assay cutoffs mandated by workplace drug testing programs are not necessarily appropriate in a clinical setting. A true positive result indicates drug use, but it cannot be used to infer impairment or intoxication of the patient (7). In addition, most screening assays are not specific for single drugs; rather, they detect drugs of a particular class with varying degrees of cross-reactivity to the drugs and their metabolites, potentially leading to false positive results (8). If a definitive result is required, analysis should be confirmed by an alternate method, for which the gold standard is gas chromatography–mass spectrometry (GC/MS) (9).

Drug analysis by GC/MS identifies and quantifies drugs at levels below those attained in screening assays and is considered the method of choice for identifying drugs or metabolites in urine (8). Quantitation of drugs by GC/MS is a labor-intensive process and not all drugs of a particular class will necessarily be identified. Therefore, when reflexing to a GC/MS confirmation assay, it is important to know which drugs the assay can identify and to understand that not every drug from a given class will necessarily be included.

Understanding Specific Drug Classes

As mentioned previously, most screening assays do not detect specific drugs. To effectively communicate the results of DAU screening assays to physicians, laboratorians need to understand how cross-reactivity affects test results for the commonly abused drugs.

Benzodiazepines are one of the most widely prescribed classes of drugs. They are used therapeutically as anxiolytics, sedative-hypnotics, anticonvulsants, and as a treatment for obsessive-compulsive disorder (
10). Along with their sedative effects, they also may cause cognitive impairment. Adverse reactions observed in pediatric and elderly populations include increased agitation and insomnia (10–12). Chronic benzodiazepine use poses a risk for the development of dependence and abuse (13), particularly use of those agents with the shortest half-life, the highest potency (e.g., alprazolam, triazolam), and greatest lipophilicity (e.g., diazepam) (14).

Benzodiazepines share some structural similarity but differ significantly in their pharmacokinetic parameters (e.g., onset of effect, half-life, volume of distribution) (10-12). They are extensively metabolized in the liver by cytochrome P450 (CYP) enzymes, particularly CYP3A4 and CYP2C19. These phase I reactions include hydroxylation, dealkylation, deamination, and reduction. Table 1 outlines the major phase I benzodiazepine metabolites (10). Following these reactions, conjugation with glucuronic acid occurs and these glucuronidated metabolites comprise the major urinary products of benzodiazepines (10–15).

The cross-reactivity in screening immunoassays of the various benzodiazepines and their metabolites varies considerably from manufacturer to manufacturer, and screening assays cannot distinguish between the individual benzodiazepines. Most of the assays are calibrated to the common metabolites oxazepam, temazepam, or nordiazepam (16). However, the large number of different functional groups that may be present on the benzodiazepine nucleus make it difficult to detect all drugs in this class, and some compounds such as midazolam, chlordiazepoxide, and flunitrazepam may not be detected by many assays (17, 18).

Table 1
Major Phase I Metabolites of Benzodiazepine
Parent Drug
Phase I Metabolites
Diazepam Nordiazepam
Chlorazepate Nordiazepam
Chlordiazepoxide Nordiazepam
Alprazolam α-Hydroxy-Alprazolam
Triazolam α-Hydroxy-Triazolam
Clonazepam 7-Amino-Clonazepam
Flunitrazepam 7-Amino-Flunitrazepam
Flurazepam Hydroxy-Ethyl-Flurazepam

Opiates comprise the naturally occurring or semisynthetic analgesic alkaloids derived from opium, the dried juice from the seeds of the poppy plant, Papaver somniferum (
9). Morphine, the principal alkaloid derived from opium, is the building block for many of the semisynthetic opioid analgesics including heroin, oxycodone, oxymorphone, hydrocodone, hydromorphone, and levorphanol (3). Opium also contains small amounts of codeine. In addition to their analgesic properties, opiates may also cause sedation, euphoria, and respiratory depression (9). Due to these euphorigenic and analgesic properties, opiates have a high abuse potential. Long-term chronic use can lead to tolerance and both physical and psychological dependence (8). Pain management programs often use urine drug testing to monitor compliance, diversion, or substitution of prescribed drugs (9). As a result, it is important to note that not every member of the opiate family of drugs can be reliably detected by an immunoassay screen for opiates.

In general, the majority of opiate screening assays target morphine with varying levels of cross-reactivity toward codeine, hydrocodone, and hydromorphone (19–21). Oxycodone, oxymorphone, and meperidine demonstrate low cross-reactivity and cannot be reliably detected by the majority of opiate screening assays. Methadone is not detected by opiate screening assays and an assay designed solely for the identification of this drug should be used when identification is required. GC/MS confirmation assays do offer quantitation of codeine, morphine, hydrocodone, and hydromorphone, as well as oxycodone and oxymorphone. For meperidine, a mass spectrometry assay specific for its quantitation is recommended.

Methadone (Dolophine) is a synthetic opioid, a compound that is structurally unrelated to the natural opiates but is capable of binding to opioid receptors. These receptor interactions create many of the same effects as seen with natural opiates, including analgesia and sedation. However, methadone does not produce feelings of euphoria and has substantially fewer withdrawal symptoms than opiates such as heroin (
10). Methadone is used clinically to relieve pain, treat opioid abstinence syndrome, and treat heroin addiction.

Patients who are taking methadone for therapeutic purposes excrete both the parent drug, methadone, and its major metabolite, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), in their urine. Clinically, it is important to measure levels of both methadone and EDDP. Methadone levels in urine are widely variable depending on factors such as dose, metabolism, and urine pH, while EDDP levels are relatively unaffected by the influence of pH and are therefore preferable for assessing compliance with therapy (15). Patients who are known to be compliant with their methadone therapy have ratios of EDDP:methadone greater than 0.60 (22). Some patients undergoing treatment with methadone have attempted to pass compliance testing by adding a portion of the supplied methadone to the urine (23). This is commonly referred to as “spiking.” In these situations the sample will contain large amounts of methadone and little or no EDDP (23, 24). In these circumstances the ratio of EDDP:methadone is less than 0.090 and strongly suggests manipulation of the urine sample (23).

Immunoassays that detect methadone do not detect EDDP, and those that detect EDDP do not detect methadone. Therefore, identification and quantitation by a confirmatory method such as GC/MS is essential.

The term amphetamine refers not only to the illicit substances amphetamine and methamphetamine but also to several chemically related phenylethylamines that are referred to collectively as the amphetamine-type stimulants (ATS). Amphetamines are sympathomimetic amines that stimulate the central nervous system and, in part, suppress appetite. In the past, many people who used legitimately prescribed amphetamine for appetite control or depression also became unwittingly habituated to its effects, leading to its classification as a Schedule II controlled substance (
15). Today, the use of amphetamine for the treatment of depression has virtually ceased and it is currently used to treat narcolepsy, attention deficit and related disorders, and minimal brain dysfunction. Although there recently has been a moderate resurgence of its illicit use, the overall clinical use of amphetamines has declined, and as such clandestine laboratories are now the major source for most of the illicit amphetamine and methamphetamine in North America as both substances are relatively easy to synthesize.

Most amphetamine immunoassays are designed to detect amphetamine/methamphetamine, while some are designed to include MDMA and MDA and, more broadly, capture the ATS group. Drugs commonly placed in the category of illicit “amphetamines” include the D-isomers of amphetamine, methamphetamine, phentermine, and the designer amines, methylenedioxymethamphetamine (Ecstasy, MDMA) and its metabolite methylenedioxyamphetamine (MDA). Unfortunately, many of the over-the-counter sympathomimetic amines present in cold medications (ephedrine, pseudoephedrine, phenylpropanolamine, and phenylephrine (7)) have been shown to produce positive screening results; additionally, many psychotropic medications have been reported to interfere with the immunoassays (15). Regardless of the immunoassay used, confirmation by a second confirmatory technique is recommended.

Marijuana and other psychoactive products obtained from the plant Cannabis sativa are the most widely used illicit drugs in the world (
15). Cannabis has been used for its euphoric effects for more that 4,000 years. Cannabinoids are a unique subset of chemicals found in the cannabis plant that have mental and physical effects associated with its use. Δ-9-Tetrahydrocannabinol (THC) is the major psychoactive chemical in the cannabis plant (15).

THC undergoes rapid hydroxylation by the CYP enzyme system to form the active metabolite 11-hydroxy-THC. Subsequent oxidation of 11-hydroxy-THC produces the inactive metabolite 11-nor-Δ-9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH). THC-COOH and its glucuronide conjugate have been identified as the major end products of metabolism. THC is highly lipid soluble resulting in its concentration and prolonged retention in fat tissue (25). The continuous slow excretion of the drug and its metabolites into the urine can produce positive drug screening results for many days or even weeks after single use, while chronic ingestion can produce positive results in the urine up to 46 days after cessation of use (26).

Urine immunoassay screens are primarily designed to detect THC-COOH with cross-reactivity towards the metabolites of THC. Nonsteroidal anti-inflammatory drugs have been reported to interfere and cause false positive results for THC in some screening immunoassays (26, 27). Other agents that have been shown to cross-react with cannabinoid immunoassays include efavirenz (28, 29) and proton pump inhibitors (30). Various chromatographic methods have been published over the years for confirmation of THC-COOH in urine specimens, including gas chromatography (GC), high-performance liquid chromatography (HPLC), and thin layer chromatography (TLC). However, GC/MS is generally accepted as the confirmatory method of choice.

Cocaine is a potent CNS stimulant that results in a state of increased alertness and euphoria. The effects of cocaine are attributed to its action at the nerve synapses by blocking dopamine reuptake (
9). Cocaine also exerts effects at the presynaptic nerve terminals by blocking the reuptake of norepinephrine. The resulting sympathomimetic responses include increased blood pressure, heart rate, and body temperature. Cocaine has been used clinically as a local anesthetic and vasoconstrictor of mucous membranes in nasal surgery, but it is also widely abused (9).

Cocaine is rapidly metabolized by liver esterases to its inactive metabolites, ecgonine methyl ester and benzoylecgonine. Cocaine may also be metabolized to benzoy-lecgonine through spontaneous hydrolysis. The half-life of cocaine ranges from 0.5 to 1.5 hours, for ecgonine methyl ester from 3 to 4 hours, and for benzoylecgonine from 4 to 7 hours (9). Given the longer half-life of the inactive metabolite, screening assays for cocaine have been designed to detect benzoylecgonine (28–30). These assays have good cross-reactivity with the parent drug, cocaine, but at lower concentrations than that of the metabolite. The majority of GC/MS confirmation assays offer quantitation of the parent drug as well as its metabolite.

Know the Limits and Communicate

Laboratory testing for drugs of abuse can provide important information to the physician as an aid in diagnosis or compliance monitoring. However, it is incumbent upon laboratorians to understand the limitations of DAU testing and take the time to educate the physicians they serve.


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Lynn Cheryk, PhD, is an assistant professor in the Department of Laboratory Medicine and Pathology at the Mayo Clinic College of Medicine (Rochester, Minn.) and laboratory director of Mayo Medical Laboratories New England (Wilmington, Mass.) 




Loralie Langman, PhD, is an assistant professor in the Department of Laboratory Medicine and Pathology at the Mayo Clinic College of Medicine and Director of the Drug/Toxicology Laboratory, Division of Clinical Biochemistry and Immunology, Department of Laboratory Medicine and Pathology, Mayo Clinic (Rochester, Minn.)