Mr X has been referred to the transplant center for liver transplant evaluation secondary to end-stage liver disease of unknown etiology. His medical history is significant for previous illicit opioid use after a tragic work-related accident for which he was treated with opioids for pain management during recovery. Mr X connected with his primary care provider on illicit drug use, and he was placed on methadone to manage his chronic pain, with a drug screening contract. Upon referral to the transplant center, routine intake laboratory testing was ordered, including a urine toxicology screen, which came back negative for the presence of methadone and opiates. Seeing these results, the intake counselor consults the laboratory director of toxicology since a previous urine toxicology screen was positive for methadone; he wants to understand why the results are discordant, particularly because the patient reports no change in administration schedule.
Opioid use and abuse has reached epidemic proportions and is a major contributing factor to overdose deaths.1 Increasingly, physicians in all medical specialties need to be prepared to assist with identifying and managing patients who are actively using opioids, or who are at risk for addiction.2 Incumbent on this is an understanding of the types of drug screening available to assess for the presence of drugs, specifically opioids, and their limitations. The goal of this article is to describe the various methodologies available for opioid testing, their strengths and weaknesses, and the importance of a strong partnership with the clinical laboratories to enhance appropriate test/assay selection and test result interpretation for the patient population being served.
Overarching principles of drug testing programs
Treating physicians in consultation with laboratory service providers can opt for one of 2 approaches to drug testing and drug monitoring: (1) screening, followed by reflex confirmation using more sophisticated techniques [eg, liquid chromatography with tandem mass spectroscopy (LC-MS/MS)] or (2) direct testing by LC-MS/MS. The former approach is most prevalent due to the availability of immunoassays that have been optimized for the commonly used chemistry analyzers. The use of direct testing by LC-MS/MS within hospital-based clinical laboratories is limited owing to the sophisticated nature of the methodology and the relatively significant capital outlay; hence, confirmatory testing is largely in the purview of reference laboratories.
Although a variety of specimen types are theoretically appropriate for drug testing, including opioid testing (Table 1)3–9, urine is the specimen of choice due in large part to ease of collection, and optimal assessment for the presence of the drug of interest and its metabolites.1,2,10 Screening and confirmatory assays have been optimized for urine testing, but it is important be aware of the limitations of urine as a specimen source due to potential adulteration.4,11 Laboratory panels may include reflex assessment of urine protein and creatinine concentration as a means to monitor for adulteration of urine specimens.11
Point-of-care (POC) testing
POC testing is often an attractive consideration for patient laboratory assessment because it offers the opportunity for rapid turnaround of results in the office/clinic setting, and, thus, the ability to provide immediate management decisions and feedback to the patient. Lateral flow immunoassays are the backbone of POC test offerings for opioids, and either urine or saliva may be used as a specimen source.12,13 In general, for POC assays, the detection of drug is based on the principle of competitive binding in which the presence of drug in the specimen competes for antibody that otherwise interacts with drug that is imbedded in the system (Fig. 1).12 If no drug is present, a colored band develops; if drug is present, no colored band develops. It is important to understand that POC test results are qualitative, identifying the presence or absence of classes of drugs (eg, opiates, amphetamines, benzodiazepines); they do not identify the presence or absence of a specific drug. Thus, if the screen is positive, the result should be considered “presumptively positive” for the drug class. Confirmatory testing through LC-MS/MS should be considered in these situations, particularly if results are unexpected.
Depending on the configuration, some POC immunoassays test for opiates (eg, morphine), in addition to amphetamines and other drugs of abuse, but do not assay for synthetic opioids (eg, methadone or buprenorphine). Thus, when setting up a drug-screening program at the POC, multiple assays may be required depending on the target population being screened.10 It is also important to understand that concentration thresholds for a positive result vary depending on the manufacturer assay used. Consequently, the end user needs to be aware that a negative result does not necessarily mean absence of the drug. Equally important, the end user of POC needs to have a clear understanding of when specimens should be forwarded to a reference/main laboratory for confirmatory testing.
Hospital laboratory testing for opiates
Similar to POC assays, immunoassays are a widely used methodology for detecting opiate compounds in hospital-based clinical laboratories due to their rapid turnaround time and capacity to detect multiple types of opiates.1,11 The use of automated chemistry analyzers for these tests is cost-effective and enables rapid results to be provided continuously, which can be critical for timely patient management, and particularly important in cases where drug overdose is suspected.14 These drug-screening tests can provide presumptive results that are integrated with more definitive mass spectrometry (MS)-based testing if needed.15 As noted earlier, urine is a specimen of choice in many clinical settings because compounds are concentrated and can have a longer detection window relative to body fluids such as serum or plasma (Table 2). The downside of urine testing is that direct observation of sample collection is less commonly performed and adulteration can readily confound interpretation.14 Additional measurements such as creatinine, specific gravity, pH, and temperature and alternative specimen types can be useful in providing further clarity in situations where specimen tampering is suspected.10
Commercial immunoassays for opiates commonly target morphine because assay development has been driven to detect illicit heroin use. Unfortunately, semisynthetic or synthetic opioids such as oxycodone, fentanyl, or methadone that are commonly encountered in modern clinical practice are variably cross-reactive using these screening tests; therefore, more specific immunoassays or MS may be a preferred testing methodology.16
Pain management clinics may require more sophisticated testing beyond general opiate immunoassays for several reasons. First, it may be important to have greater assay sensitivity than many of the commonly performed urine drug screen tests can provide. Second, the quantification of metabolites and detection of synthetic opioids may be critical for monitoring compliance with prescription pain medications to prevent substance abuse.4 Therefore, it is important to balance the clinical need for rapid turnaround time, detection limits, and accuracy in a hospital setting that provides services for emergent care and outpatient clinics when evaluating optimal testing methodologies.
One of the most commonly used techniques for urine drug screening is the competitive immunoassay. In most of these assays, metabolites in a patient specimen are incubated in the presence of a known quantity of labeled drug, with labeled and unlabeled drug competing with each other for antibody binding.14 The drug label and detection method can vary widely between different manufacturers and moieties that enable spectrophotometric, enzymatic, fluorescent, chemiluminescent, and other detection methods are commonly incorporated. Results are usually reported qualitatively on the basis of a calibrated cutoff value, a threshold that is often set at 300 ng/mL for many urine opiate tests.14,16
These competitive immunoassays can be further classified by assay design and detection methodology. Commonly used strategies for drugs of abuse testing include kinetic interaction of microparticles in solution (KIMS), cloned enzyme donor immunoassays (CEDIA), and enzyme multiplied immunoassay technique (EMIT) (Fig. 2).14,16–20 Techniques that incorporate the use of radioimmunoassay and fluorescence polarization have also been applied to drugs of abuse testing21; however, these assay designs are less commonly used in current laboratory practice.
KIMS assays measure the changes in light absorbance that occur when drug conjugates that are bound to microparticles are incubated with anti-drug antibodies in the presence of a patient sample. If there is no drug present in a patient’s urine, the drug-conjugated microparticles bind the anti-drug antibodies to form aggregates that block light transmission. Therefore, a negative test result has increased light absorbance. However, if the patient’s specimen does contain drug, then competition for antibody-binding sites occurs and the drug-conjugated microparticles cannot readily access the antibody molecules, causing a decrease in light absorbance. When the absorbance reaches the calibrated threshold level, the specimen is interpreted as positive.14,20
The technology underlying CEDIA assays harnesses the use of 2 separate fragments of the β-galactosidase protein that can recombine to restore its enzymatic activity. In this assay, the drug is attached to a fragment of the β-galactosidase enzyme called the enzyme donor (ED), which is incubated with the patient’s sample in the presence of an enzyme acceptor fragment of the β-galactosidase enzyme and anti-drug antibodies. If there is no drug present in the patient’s sample, then the ED cannot bind to the enzyme acceptor to form an active β-galactosidase enzyme. However, if there is drug present in the patient’s sample, then this unlabeled drug competes with the drug attached to the ED for binding to the anti-drug antibodies, and active β-galactosidase enzyme is formed. Therefore, more β-galactosidase enzymatic activity would be detected in a patient sample that is positive for the drug and the amount of active enzyme present is monitored by hydrolysis of a colorimetric substrate.14,18
EMIT assays are another variation on this strategy, with drug in the patient’s sample competing with drug that has been labeled with glucose-6-phosphate dehydrogenase (G6PDH) for binding with anti-drug antibodies. If the patient’s sample does not contain any drug, then the labeled drug binds the antibody, blocking the enzymatic activity of G6PDH. In this situation, the cofactor nicotinamide adenine dinucleotide (NAD) is not converted into its reduced form (NADH) and there is no change in the absorbance of the reaction mixture. However, if drug is present in the patient’s sample, then it can compete for antibody-binding sites, allowing G6PDH activity to generate NADH from NAD, creating a change in absorbance that can be detected spectrophotometrically.14,20 All of these test methodologies are subject to false negatives and false positives depending on the avidity, affinity, and specificity of the antibodies used in the immunoassays and patient factors that can impact test results.
Interpretation of clinical results
Factors that can influence the detection of drugs of abuse in the urine include the amount and purity of the drug ingested, the frequency of drug use, body mass, pharmacokinetics, assay threshold, and timing of the sample collection. Common causes for false negatives include lack of compound cross-reactivity and insufficient sensitivity of the assay methodology, sample collection outside of the temporal detection window for the test, and issues with specimen quality due to adulteration or tampering (Table 2).4,11 In these cases, MS testing, alternate sample matrices, or repeat collection with direct observation may clarify the initial clinical testing results.
Potential sources of false-positive results from urine opiate screening tests include the patient’s diet and ingestion of over-the-counter and prescription medications. Consumption of poppy seed-containing foods has been shown to cause positive urine immunoassay results in a number of studies,22,23 which has in turn impacted the cutoffs for workplace testing.11 In December 1998, opiate cutoffs were increased from 300 to 2000 ng/mL to avoid false positives from poppy seed consumption by the Department of Health and Human Services; however, the lower cutoff is frequently used in clinical laboratories to detect the use of opiates by patients.11 Dextromorphan, a common ingredient in over-the-counter cough medications, has been associated with false-positive urine opiate testing24 and multiple reports of prescription medications including quinolone antibiotics25,26 and rifampin27 have been documented to cause false-positive urine screens. Therefore, it is of utmost importance to perform a thorough patient interview, reviewing both prescription and over-the-counter medications, because any information elicited can help with interpretation of unexpected results.
Specimen adulteration and alternative sample matrices
The color, appearance, and temperature of a patient’s urine immediately after sample collection can be useful to evaluate whether the specimen may have been adulterated or substituted. Extremely high or low specific gravity (outside of the range of 1.002 to 1.020) or pH values (outside of the range of 3 to 11) can be indicative of specimen contamination. Extremes of temperature (beyond 32 to 38°C) or low urinary creatinine (below 20 mg/dL) can also suggest adulteration. Unfortunately, these additional measurements to evaluate patient samples are not routinely performed on all clinical specimens and ordering providers may not always request that these tests be performed after a urine specimen collection.4,11
If urine specimen tampering is suspected, then evaluation using an alternate test methodology or a different sample matrix may be warranted. Blood testing can be performed and oral fluid measurements can also be assessed, which may be advantageous as both of these sample types are directly collected from the patient under observation. Basic drugs including oxycodone, hydrocodone, buprenorphine, fentanyl, and methadone are present at higher concentrations in saliva relative to blood due to pH differences between these body fluids. However, the disadvantages for oral fluid testing relative to conventional urine drug screening are that the window period for drug recovery is shorter in saliva relative to urine and the specimen volume is limited, which can also make the detection of illicit drugs more challenging.4,10 Drug testing in hair is not frequently used as specimen preparation is costly and time-consuming, and results can be confounded by coloration and environmental contamination.4
Opioid confirmatory testing
As discussed previously, most presumptive positive results should be confirmed by a definitive method such as MS. Compared with immunoassays, MS-based methods provide several key advantages. They allow definitive identification of opioids and metabolites, have much lower limits of detection, and are compatible with many sample types (urine, serum, oral fluid). These analytical features are important in reconciling unexpected immunoassay results (positive or negative), determining if a patient has diverted their prescription or has adulterated their sample to “pass the test,” or is abusing a nonprescribed opioid.
Confirmatory testing is especially important for opioids, which comprise 6 chemically distinct classes that are not equally recognized by immunoassays.28 The most familiar class of the opioids, the phenanthrenes, are related by a basic morphine structure and, among others, includes codeine, morphine, and heroin. In contrast, other opioid classes have significant modifications (methadone) or are structurally unrelated (fentanyl). Therefore, opiate immunoassays using morphine as the antigenic target will either fail to detect, or will have invariably low cross-reactivity with classes of opioids commonly used in pain management and illicit abuse.29
The workflow of confirmatory testing involves sample preparation, chromatography, and MS. For toxicology and drug testing, chromatography was historically performed with either gas chromatography (GC) or liquid chromatography (LC). Although GC is efficient and provides excellent chromatographic resolution, a major disadvantage is that it requires compounds to be volatile. Many opioids require chemical derivatization to produce compounds that are sufficiently volatile for GC, whereas LC methods do not require this time-consuming step.28 Consequently, LC combined with tandem MS has largely replaced GC-MS as the gold standard for opioid confirmatory testing.30
Sample preparation and chromatography
The matrix of clinical specimens is complex and sample preparation is an essential step before chromatography. Sample preparation provides a cleaner sample, removes interfering substances, and can concentrate the desired analyte. The simplest method for preparing samples is the “dilute and shoot” technique in which the sample is directly mixed with buffer and internal standard and injected into the chromatographic system.31 Other methods of sample preparation are more extensive and include protein precipitation, hydrolysis, derivatization, liquid-liquid extraction, and solid-phase extraction.32
Once the sample has been adequately prepared, the analyte of interest can be purified using LC. Chromatography exploits the biophysical differences that exist between unlike molecules and how they differentially distribute between the stationary and mobile phases.33 In LC, the analytical column is the stationary phase and the buffer is the mobile phase. Opioid analysis uses reverse-phase chromatography, a type of chromatography that combines a nonpolar stationary phase with a polar mobile phase. Typically, a nonpolar column such as C-18, biphenyl, or pentafluorophenyl is combined with a polar mobile phase such as acetonitrile, methanol, and water.34 The sample can be directly introduced into the LC system by an injection loop and carried onto the analytical column through the action of pumps and buffer. To optimize resolution and efficiency of chromatographic separation, the “strength” of the mobile phase can be held at a constant (isocratic) or increased gradually over time (gradient). LC systems are commonly interfaced with mass spectrometers to provide a means of detection, forming “hyphenated” methods such as LC-MS. Reverse-phase LC-MS is the method of choice for opioid quantification and can achieve detection limits as low as 0.5 ng/mL.28
The mass spectrometer
In a basic sense, a mass spectrometer distinguishes molecules by their mass (or more accurately mass-to-charge ratio, m/z). This enables the identification of parent molecules and/or fragments that are characteristic of a given molecule. The major components of a mass spectrometer include a sample inlet, ionization source, a mass analyzer, and an ion detector (Fig. 3).35 The effluent from the LC system enters the mass spectrometer by a sample inlet, where it is ionized to forms charged ions, and then separated by the mass analyzer before reaching the detector.
Ionization is a necessary step that allows mass analyzers to separate and identify specific molecules. This occurs in the ionization source, where the molecule of interest becomes charged. A common and efficient method of ionization for biomolecules is electrospray ionization. 36 With electrospray ionization, effluent from the LC system passes through a capillary tip to which voltage is applied, producing a fine spray of charged droplets. The addition of heat facilitates solvent evaporation and as the size of the droplet decreases, there is a consequential increase in charge density. This inevitably causes charged ions to be expelled from the droplet and the process continues until each droplet contains a single molecule that is then directed into the mass analyzer for identification.32
The mass analyzer sorts and separates ions according to their mass-to-charge ratio (m/z). It is the heart of the mass spectrometer; performance characteristics such as the instrument’s resolution, accuracy, and range vary depending on the type of mass analyzer.36,37 A popular type of mass analyzer in clinical laboratories is the quadrupole. Quadrupole mass analyzers are widely used in clinical laboratories as they provide many attractive features, including ease of use and sufficient performance characteristics.36 Quadrupole mass analyzers consist of 4 parallel metal rods arranged in a square formation, forming a 3-dimensional channel through which ions can travel (Fig. 4).35 Only ions with a specific m/z will move through the quadrupole in a stable trajectory and reach the detector, whereas ions of a different m/z will have an unstable path and be deflected. The voltages applied function to select which ions reach the detector and which ions do not; hence, quadrupoles are also called “mass filters.” By varying the voltages, the rods can be scanned from low to high mass (full scan) or fixed to select individual m/z (selected ion monitoring). The former is good for unknown screening, whereas the latter is useful for targeted analysis.35
One feature that makes mass spectrometers extremely versatile is the various arrangements that they can assume. A common configuration is the triple quadrupole mass spectrometer, also called the tandem mass spectrometer (MS/MS). Tandem MS/MS links 3 quadrupoles together in a sequential manner. The first quadrupole scans a m/z range and selects an ion of interest, the second fragments the ions selected, and the third analyzes the product ions generated in the second quadrupole.35 This method is not only highly selective and sensitive but is also able to measure multiple analytes in a single sample. Furthermore, tandem MS/MS can be applied to carry out targeted analysis or screen for unknown analytes.35
Disadvantages of MS
MS-based methods are extremely powerful and undoubtedly a definitive method, a “gold standard” for opioid confirmation. However, they have their own set of challenges such as ion suppression and the presence of isobaric compounds. Ion suppression is a matrix effect where a component in the sample reduces the efficiency of ionization and thereby suppresses the signal. Modifying the preparation of sample or LC conditions is a helpful approach in minimizing or eliminating this type of interference.38 Compounds that have an identical molecular mass are called isobars and represent another source of interference with MS-based methods. Some opioids are isobaric and very difficult to distinguish because they also form the same fragments during ionization.31 This is the case for patients taking venlafaxine, a commonly prescribed antidepressant. These patients generate false-positive tramadol results by LC-MS/MS because the metabolites of venlafaxine are isobaric with tramadol and share the same MS/MS spectral library match.39 Careful chromatography and validation is essential in distinguishing these patients. Finally, MS is also a costly technical challenge and not easily implemented in the routine hospital laboratory.29 Capital expenses associated with initial instrumentation purchases are not easily justified, especially in light of the longer turnaround time of test results. In addition, daily operation and troubleshooting require specialized training with all steps of preparation, operation, and analysis.40,41
Appreciating both technical utility and the limitations of opioid drug screening is critical for the interpretation of laboratory testing in an era where prescription drug abuse is becoming increasingly prevalent. The clinical laboratory plays an important role in guiding ordering providers toward the appropriate interpretation of screening tests and encouraging the use of more specific confirmatory assays or alternate sample types under circumstances when false-positive or false-negative results may occur. In addition to offering guidance on specific assay results, clinical laboratories may need to adapt testing strategies to reflect the changing patterns of drug abuse seen in practice to better serve the needs of clinicians and patients within their communities.
Conflict of interest disclosure
The authors declare that they have nothing to disclose.
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