Modern drug testing in clinical laboratories: a narrative review of practical approaches and emerging technologies

Introduction

Clinical toxicology testing is an integral part of laboratory medicine to support patient care and drug management and it involves the identification and/or quantification of drugs, metabolites, trace and toxic elements, and illicit substances in biological specimens. This type of testing encompasses therapeutic drug monitoring (TDM), newborn drug testing, drug testing, and trace and toxic elements. TDM is indicated for medications with narrow therapeutic windows to optimize dosing regimens to achieve drug efficacy while minimizing the risk of toxicity. Pain management and substance use clinics definitive technology to assess adherence to patients on controlled substances and drug screens to detect unreported drug use. In the emergency department, patients may exhibit signs and symptoms of toxicity; therefore, rapid drug and toxin screening can enable life-saving interventions (1). Frequently, a poisoned patient requires an extensive workup to determine the cause of their symptoms. This may be achieved using a combination of drug screens, targeted confirmations of suspected agents, and untargeted analyses to identify an unknown cause. Testing newborns for drug exposure can expose maternal substance use during pregnancy and guide support decisions for child protection and addiction support services (2). Trace and toxic element analysis can assess nutritional deficiencies/toxicity and environmental or occupational exposures to heavy metals (3).

Toxicology testing can incorporate various methodologies, such as immunoassay and mass spectrometry. Immunoassays offer speed and minimal sample preparation for high-throughput or point-of-care (POC) use. However, these assays may lack analytical specificity and are prone to cross-reactivity to molecules that are structurally similar, which could lead to false-positive results (4,5). Mass spectrometry may provide lower sensitivity and increased specificity to detect a broad range of analytes. However, laboratories must evaluate factors, such as cost, specimen matrix for testing, sample preparation, turnaround time to result, technical expertise for maintenance, operation, and method development/validation, and clinical laboratory space, to determine if mass spectrometry will meet the clinical testing needs.

In this review, we provide a comprehensive overview of current technologies used to monitor drug use, with emphasis on recent developments and ongoing challenges in the field. We examine the advantages and limitations of commonly used specimen types, as well as the potential utility of novel matrices in toxicology testing. Both established practices and emerging approaches are considered, alongside the interpretive challenges encountered in routine practice. Finally, we outline key factors in instrument selection to guide laboratory decision-making. We present this article in accordance with the Narrative Review reporting checklist (available at https://jlpm.amegroups.org/article/view/10.21037/jlpm-25-23/rc).

Methods

This narrative review was conducted to summarize current practices, challenges, and future directions in clinical toxicology testing. Relevant literature was identified through selective searches of PubMed and key laboratory medicine journals, with a focus on peer reviewed publications from the past 10–15 years written in or translated to English. Key journals accessed include the Journal of Analytical Toxicology, Journal of Applied Laboratory Medicine, Clinical Chemistry, Clinical Chemistry and Laboratory Medicine, and Clinics in Laboratory Medicine. Additional sources included guideline documents from professional societies [e.g., College of American Pathologists (CAP), Association for Diagnostics & Laboratory Medicine (ADLM)] and authoritative textbooks. These sources were referenced between January and September 2025. The review prioritized studies and reviews addressing specimen selection, analytical methodologies (immunoassay, mass spectrometry), interpretation strategies, and technological innovations (Table 1). Articles were selected based on clinical relevance and expert consensus, rather than exhaustive coverage of all available data.

Toxicology testing considerations

Specimen types

The choice of specimen type in toxicology testing depends on factors such as detection window, ease of collection, and susceptibility to adulteration. Additionally, whether the parent drug or its metabolite predominates in a given specimen determines the availability of appropriate analytical standards. These considerations underscore the need to align specimen selection with both clinical context and laboratory feasibility.

Urine

Urine has historically been the preferred sample type for toxicology testing due to its ease of collection (no needlestick required) and extended detection window relative to blood (6,7). Urine requires simpler sample preparation protocols compared to blood prior to analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (8). While urine is a useful sample type for toxicology, this matrix does have several limitations. First, drug concentration in urine does not reflect pharmacodynamics and can be influenced by factors such as hydration status, pharmacogenetics, liver disease, or kidney dysfunction (9). This limits the utility of urine in trending drug levels over time. Second, the collection of urine cannot be witnessed without an invasion of privacy. Finally, this matrix is particularly susceptible to tampering due to the ease of adulteration. Urine may be substituted for an alternative specimen or altered via dilution or the addition of substances with the goal of passing a drug test. For example, nitrite-containing adulterants can oxidize the delta-9-tetrahydrocannabinol (THC) acid metabolite, rendering the levels undetectable for confirmation testing (9).

Blood

Blood as a matrix for toxicological assays has the benefit of direct collection, so the risk for adulteration is much lower. In addition, drug concentrations in blood correlate better with pharmacodynamics, which is why it is often the preferred matrix for TDM (9). The drawbacks to using blood include the need for a needlestick and a reduced detection window compared to urine. For example, the window of detection for benzoylecgonine in the blood is measured in hours, whereas it can be detected in urine for 1–3 days (or longer for chronic users) (10). Analytically, blood often requires more extensive sample cleanup than urine, since lipids, cellular debris, and other matrix components must be removed prior to analysis. This may include costs for both supplies and technologist time.

Oral fluid

Oral fluid has recently been gaining popularity as a matrix because it is easy to collect and, unlike with urine, the collection can be directly witnessed without the infringement of privacy. Its ease of collection makes it an appealing option for POC and in-the-field testing. However, the slightly acidic nature of oral fluid may lead to ion trapping and higher concentrations of basic, non-protein bound drugs compared to blood (11). Conversely, acidic drugs are frequently found at lower concentrations compared to blood. Oral fluid exhibits lower concentrations and shorter windows of detection when compared to urine, often limiting its use to very recent exposure (12). The shorter detection window may be desirable for certain analytes, such as THCCOOH (11-nor-9-carboxy-THC), which can persist in urine for weeks in chronic users (13). It may also be useful for monitoring adherence to prescribed medications taken daily, such as buprenorphine.

Data on the interpretation of drugs and their metabolites in oral fluid is growing, but still limited, and several factors confound result interpretation. For example, parent drugs often are detected more readily than metabolites, which contrasts with urine and blood, where metabolites typically predominate. Oral fluid is particularly susceptible to contamination by environmental compounds (e.g., smoke or uncoated drugs) (11). Finally, the route of administration (e.g., inhalation that coats the oral cavity vs. orally consumed encapsulated drug) drastically impacts the concentration that will be detected in oral fluid (12).

Several factors make oral fluid a challenging matrix to analyze. First, there are several different collection techniques ranging from commercially available collection devices to direct salivary collection, with or without the use of stimuli. Collection may be hindered by various medical conditions and specific drug use, such as cannabis, that result in dry mouth. Combined, these factors can lead to differences in protein content, viscosity, and pH at the collection step alone (11,12). Second, the limited sample volume and stabilizing buffer found in collection devices present analytical challenges (14). The surfactants, preservatives, and dyes require more complex sample cleanup methods, leading to matrix effects, and reducing the lifespan of a column (15-17). Finally, the variation posed by the collection and analysis of oral fluid makes it challenging to determine standardized detection limits like we see with urine. This makes it difficult to confidently provide detection windows after drug use. The utility of oral fluid may be limited to monitoring compliance with drugs that are readily detected in oral fluid and are expected to be taken at least daily.

Alternative matrices

Urine and blood (whole blood, serum, or plasma) remain the primary specimens used in toxicology and TDM, respectively, but alternative biological matrices are occasionally used depending on the clinical or forensic context. Dried blood spots (DBS) are a matrix that is beneficial for remote specimen collection and is clinically used for newborn screening of metabolic disorders; however, this matrix has clinical applications for clinical chemistry, TDM, infectious disease monitoring, epidemiological studies and forensics (18-20). The benefits of using DBS are that it is minimally invasive for microsampling, it can enhance the stability of the analyte, and reduce the biohazard risk for specimen transport (21). The limitations for widespread implementation of DBS for clinical practice are: analyte concentrations can differ between whole blood and serum/plasma, which could impact the adoption of the reference range or therapeutic/toxic range for all three matrices. Hematocrit can impact the volume of blood collected on a DBS card. Hematocrit can impact the volume of blood collected on a DBS card. Validated methods for DBS are currently not available for every analyte, and there are limited guidelines for DBS quality control (QC) samples and method validation experiments for clinical testing (21).

Unique to the neonatal population, umbilical cord tissue and meconium may be used to determine in utero drug exposure. Meconium represents drug exposure during the second and third trimester and provides greater sensitivity than the umbilical cord, but the collection of meconium is challenging and may take several days to be passed (22). Umbilical cord, on the other hand, only encompasses drug exposure during the third trimester, but it can easily be collected immediately after birth. Neonatal matrices show considerable variability when used to interpret the rough timing of drug exposure and may test positive for drugs administered during labor and delivery (23). Breast milk is not routinely tested in the clinical lab, but it may be a useful specimen if trying to determine the risk of transmission to an infant. For long-term exposure, hair is the matrix of choice. Drugs may incorporate into hair via the blood supply, sweat, or external contamination and is stable in the hair under most conditions. However, dyes, bleach, and other hair treatments may strip drugs from hair while also making hair more susceptible to external contamination (24). In addition, hair pigment is directly correlated to how tightly drug binds to hair, with darker hair exhibiting a higher concentration of the drug compared to lighter hair (25). As such, the use of hair for drug testing should be interpreted with caution, keeping potential racial biases in mind, and only used in select cases. Hair is also a challenging matrix to analyze due to the complexity of analytical steps required to prepare this matrix for testing, including decontamination to remove environmental contamination (24).

Future matrices

The field of clinical toxicology continues to evolve, including advancements in specimen matrices. Recent efforts have focused on developing breath-based assays for rapid and accurate detection of cannabis, with applications spanning clinical, forensic, and anti-doping contexts (26-30). Breath collection is noninvasive and requires minimal pre-analytical processing, though challenges remain in optimizing collection devices, understanding pharmacokinetics, and improving analytical sensitivity. Fingerprint sweat is another novel matrix under investigation, offering noninvasive and rapid sample collection. Early studies have demonstrated the feasibility of detecting drugs such as cocaine, THC, opiates, and amphetamines with good concordance to traditional matrices, supporting their potential role in both clinical and forensic settings (31-33). As the demand for rapid, noninvasive, and context-appropriate drug testing grows, continued development and validation of alternative matrices will be critical to expanding the clinical utility of toxicology assays.

Screening technologies

Urine drug screens are a common part of an initial patient workup, particularly in settings such as pain management, addiction medicine, and the emergency department. In larger hospital settings, screens are most commonly done by immunoassay, but POC lateral flow assays are a popular alternative in smaller clinics. These screening technologies, however, are prone to both false positive and false negative results, so more sensitive and specific alternatives are increasingly being explored.

POC screening

The method principle of a POC drug screen cup is a competitive immunoassay between the drug that may be present in urine and the drug-protein conjugate reagent on the test strip (34). The cup may use nitrocellulose strip membranes that are coated with the drug-protein conjugate and a pad that contains the colored antibody conjugate. When urine fills the cup, lateral flow is used for the urine to travel up to the antibody conjugate, then to the immobile drug-protein in the test region. When the patient’s urine is negative for the drug or the drug is below the cutoff of the assay, the colored antibody conjugate and the immobilized drug-protein will bind together and form a colored line in the test region. If there is drug present in the patient’s urine, there will be competition between the drug-protein and the drug for antibody sites on the test strip. The control line indicates that the test was performed correctly. A negative result will have two lines present, one in the test and control areas. A positive result will have only one line in the control area (35). POC drug screen cups afford rapid drug screen results within 5–10 mins of specimen collection.

Enzyme and immunoassay-based screening

Immunoassay drug screens rely on a variety of techniques including cloned enzyme donor immunoassay, fluorescence polarization immunoassay (FPIA), and enzyme-linked immunosorbent assay (ELISA) (14). Two of the most commonly employed techniques are the kinetic interaction of microparticles in solution (KIMS) and enzyme-multiplied immunoassay technique (EMIT). In a KIMS assay, free antibody binds competitively to either drug-microparticle conjugates or endogenous drug. In the absence of drug, the antibodies bind to the drug-microparticle conjugates, forming particle aggregates and increasing absorbance. When a drug is present in the sample, it competes with the conjugated drug for antibody binding. The greater the concentration of endogenous drug, the fewer antibodies bind to the microparticle-conjugated drug, resulting in less particle aggregation and decreased absorbance. Thus, drug concentration is inversely proportional to absorbance (14). In contrast, EMIT involves competitive binding between endogenous drug and glucose-6-phosphate dehydrogenase (G6PD)-labeled drug for free antibody. In the absence of endogenous drug, antibodies bind to the G6PD-labeled drug, inhibiting enzyme activity and preventing signal generation. When an endogenous drug is present, it competes with the labeled drug for antibody binding. The more antibody that binds to endogenous drug, the more unbound G6PD-labeled drug remains, allowing for enzymatic activity. Therefore, enzyme activity is directly proportional to the concentration of endogenous drug in the sample (36).

It is well documented that a major limitation of urine drug screens is the high false positive rate due to cross reactivity with similarly structured compounds (5,37-41). A well-known example is a presumptive positive fentanyl screen due to labetalol, used for treatment of hypertensive disease in pregnancy, which can lead to misinterpretations with significant consequences (42). Efforts by vendors have been made to minimize the false positive rates with newer generations of each assay. For example, the ARK fentanyl assay exhibited 75% concordance with LC-MS/MS, whereas the ARK II assay had 93% concordance due to improved specificity (43). To minimize false positives, laboratories are advised to discontinue urine drug screens for any drugs with a low prevalence, especially if they have a high false positive rate (4). A common example for this is the discontinuation of phencyclidine (PCP) drug screening for many laboratories due to its low prevalence and the high false positive rates due to commonly prescribed drugs such as venlafaxine and lamotrigine (44-47).

False negative results are also commonly observed due to poor cross reactivity with all drugs within a particular class or high cutoff concentrations. Many benzodiazepine screens do not cross react well with glucuronidated metabolites of commonly prescribed medications, such as lorazepam, and frequently result in false negatives. Recent improvements in one benzodiazepine immunoassay includes the addition of a glucuronidase to improve the clinical sensitivity from <70% in older generation assays to 95–100% in the improved assay (48). Another example of minimizing false negatives can be seen with fentanyl immunoassays; newer generation assays have improved sensitivity due to the detection of norfentanyl (49). The utility of the urine drug screen will continue to improve as manufacturers address these issues with newer generations of each assay.

Mass spectrometry-based screening

A major limitation of these drug screens, in addition to the high rate of false results, is the inability to pick up novel drugs as they arise, including fentanyl analogs, synthetic cannabinoids, and xylazine. Some labs have overcome this limitation by using advanced technology such as mass spectrometry to ‘screen’ for drugs. While some labs use targeted LC-MS/MS assays to detect a large number of drugs (50), many others use untargeted analysis through high-resolution MS analyzers to qualitatively report drug detection (51-53). These are often described as ‘screens’ because they are qualitative, but given the reliability of the technology, they are more akin to ‘qualitative confirmations’ instead. The benefits of these methods include a single workflow to detect all drugs and eliminating the need to run a single sample on multiple different assays or platforms. In addition, using a single method is easier for staff, eliminates workflow challenges (e.g., how to get samples that screened positive to the mass spectrometry lab), and only requires a single set of laboratory information system (LIS) rules. Unfortunately, using these to replace immunoassay screens increases turnaround time and requires a more skilled technical staff.

Confirmation technologies

It is generally recommended to confirm any sample that screens positive using a definitive technology. Due to its sensitive and specific nature, mass spectrometry is the assay of choice. This may be coupled to gas chromatography or liquid chromatography, and a variety of detectors may be used (54).

Gas chromatography-mass spectrometry (GC-MS)

GC-MS has been widely used in the fields of toxicology, environmental, and pharmaceutical industries for decades and offers high specificity for molecules that are volatile and can withstand temperatures up to 450 ℃. GC-MS provides compound identification based on mass spectral data from commercial or custom spectral libraries and retention time on the analytical column (36). For molecules that are not volatile, they can become chemically modified through derivatization to enhance its ability to evaporate for analytical detection. An aliquot of specimen is introduced into the GC-MS through a head chromatographic column, containing a stationary phase and an inert carrier gas (e.g., helium) (36). When the compounds exit the column, they enter the mass spectrometer, where the compounds become charged through electron impact or chemical ionization, and detected by their mass-to-charge (m/z) ratio. Ionization of molecules can also occur from positive and negative chemical ionization (36). Molecules that interact with the stationary phase will have a longer retention time before reaching the detector, and the identity of the molecules is based on retention time and mass spectral data (55,56). Drug screening by GC-MS can be performed using commercially available libraries, using electron-impact mass spectral databases (e.g., National Institute of Standards and Technology library, Wiley Registry of Mass Spectral Data), or the end-user can create a personalized database.

LC-MS/MS

LC-MS/MS has become a leading technology in clinical toxicology testing. It offers several benefits over GC-MS, such as higher sensitivity, quicker analysis times, and the ability to examine a wider range of compounds, including those that are non-volatile. The tandem mass spectrometry component allows for more accurate identification and quantification of analytes through multiple stages of mass analysis (36). This technology is particularly effective for detecting drugs of abuse, therapeutic drugs, and naturally occurring compounds in biological samples.

Liquid chromatography (LC) is a method used to separate analytes in a mixture based on their different distributions between a stationary phase (solid or liquid immobilized on a solid support within a column) and a mobile phase (a solvent or solvent mixture that flows through the column) (36). High-performance liquid chromatography (HPLC) involves using a pump to deliver the mobile phase at a constant flow rate and injecting the sample onto the chromatographic column under positive pressure. Factors such as the type of stationary phase, composition of the mobile phase, flow rate, and temperature play crucial roles in the separation of analytes. Due to its compatibility with automation and consistent reproducibility, HPLC is one of the most commonly used LC techniques in LC-MS applications, both in medical laboratories and analytical chemistry. Variations of conventional HPLC include low-flow (nano or micro) HPLC and ultra-high performance liquid chromatography (UHPLC).

When paired with an LC system, the mass spectrometer acts as an ion detector. The analyte is charged (ionized) in the source of the mass spectrometer through various mechanisms (57). Once ionized, the analyte ions move to the mass analyzer component, where they are selected based on their m/z ratio. Ions from small molecule analytes (less than 1,000 Da) usually have a single charge, making their m/z equivalent to the mass of the ion. This is often represented as [M + H]+ for protonated (positive) ions and [M – H]− for deprotonated (negative) ions. Larger molecules, like peptides and proteins, can have multiple charges, so their m/z is the mass of the ion divided by the number of charges. The selected ions then travel to the detector, and the data output is shown as a mass spectrum, with m/z on the x-axis and signal abundance on the y-axis. When combined with an LC system, a chromatogram can be generated, plotting the relative ion abundance of specific m/z values over time (36). LC-MS/MS has several advantages, such as higher sensitivity, faster turnaround time to result, and the detection of a wider range of compounds, in comparison to GC-MS.

Time-of-flight mass spectrometry (TOF-MS)

TOF-MS measures the m/z ratio of ions that are accelerated by an electric field. The ions travel through a flight tube, and the ions with a lower mass are accelerated to the detector before higher mass molecules, which take a longer time to reach the detector (36). The identity of the molecules is based on the m/z ratio and retention time. The benefits of TOF-MS are that the technique acquires full mass spectra, allowing for rapid analysis. The detector can analyze a range of masses from small to large molecules and it has high sensitivity to detect molecules of low abundance. The limitations of TOF-MS include lower resolution compared to Orbitrap, complex data interpretation, and high capital equipment expenditure.

The quadrupole time-of-flight (QTOF) mass spectrometer adds another level of specificity by filtering ions with the m/z of interest through the quadrupole, then the ions undergo collision-induced dissociation to fragment the parent ion into product ions. The product ions are accelerated into the TOF tube, where they are separated based on their m/z ratio. The technological advancements of QTOF are high-resolution mass spectra, mass accuracy, high analytical sensitivity, and the ability to provide qualitative and quantitative results. The limitations of QTOF are the high capital expense, complex data analysis, labor expenses for instrument operation, and maintenance. High-resolution mass spectrometry (HRMS) offers high sensitivity and mass accuracy to several decimal places. HRMS can acquire full mass spectra, and the strengths and limitations are similar to TOF and QTOF (58).

Future of mass spectrometry

While LC-MS/MS technology is continuously advancing, there has long been a desire for random access, automated mass spectrometry-based platforms in the clinical lab space. In 2017, the first ever U.S. Food and Drug Administration (FDA)-cleared LC-MS assay was made available for vitamin D (59). A second automated platform was made available shortly after (60), but both have since been discontinued. One of the major limitations of these platforms was the limited test menu, with both offering only vitamin D assays. Currently and over the next several years, a new automated LC-MS/MS-based assay will be introduced globally that will comprise of more than 60 analytes (61). Once the drugs of abuse are available on this platform, it will allow for a rapid turnaround time with minimal interferences, minimizing the need for a separate screen and confirmation.

The envisioned future for mass spectrometry is the reduction in cost (capital expenditure and maintenance) and footprint size to allow for analysis near the bedside of patients. There will be commercially available targeted and untargeted libraries for standardization among laboratories and NIST materials will span the drugs and molecules from the mass spectrometry libraries [cathinones and pyrovalerone derivatives (e.g., bath salts), sedative-hypnotics (e.g., designer benzodiazepines, ɣ-aminobutyric acid analogs), dissociative drugs (e.g., ketamine), psychedelics and synthetic hallucinogens (e.g., phenethylamines, psilocybin), synthetic cannabinoid receptor agonists (e.g., JWH-018), synthetic opioids (e.g., fentanyl analogs, nitazenes), stimulants (e.g., designer amphetamines), other novel psychoactive substances (NPS) (e.g., kratom), and toxic additives (e.g., xylazine, levamisole)]. Moreover, machine learning and artificial intelligence (AI) would be used to predict mass spectra (62).

The instruments will be user-friendly, similar to plug-and-play, and have interfaces to incorporate data analytics tools for data management to enable integration into hospital and clinic laboratories (63). Online, automated sample preparation would be implemented (64), not only for urine and serum/plasma, but for complex matrices such as hair, oral fluid, meconium, umbilical cord tissue, sweat, breath, etc. (65,66). The turnaround time to result will be under 10 minutes for broad-spectrum drug panels with random access to allow for STAT (immediate/urgent testing) samples. The mass spectrometry workflow would accommodate proteomics and metabolomics as routine testing in the clinical laboratory (67-69). Lastly, mass spectrometry would replace POC and immunoassay drug screens, and there would be current procedural terminology (CPT) codes to reflect new technology and newer drugs.

Considerations for screening and high-resolution methods

When evaluating toxicology platforms, laboratories must weigh acquisition and maintenance costs, staffing requirements, regulatory considerations, and technical feasibility (Table 2). Immunoassays and enzyme-based screenings generally offer lower up-front costs, minimal maintenance, and straightforward implementation, particularly when FDA-cleared kits are available. These assays require less specialized staff training and can be integrated rapidly into routine workflows, making them advantageous for smaller laboratories or those with limited technical expertise. However, recurring reagent expenses and the need for separate assays for each drug class can lead to higher per-sample costs over time, especially in high-volume settings.

By contrast, mass spectrometry-based methods require a substantial initial investment in instrumentation, service contracts, and infrastructure, along with more extensive maintenance and highly trained personnel. As laboratory-developed tests, these assays also require rigorous validation before clinical use. Despite these barriers, mass spectrometry provides broad analyte coverage, the flexibility to adapt panels as drug trends shift, and lower marginal costs per test once established. For large reference laboratories or academic medical centers with sufficient volume and expertise, mass spectrometry may offer long-term cost efficiencies and scalability that outweigh its higher entry costs. In smaller or resource-limited laboratories, immunoassays remain a pragmatic first-line approach, with confirmatory testing referred out to specialized centers.

Mass spectrometry selection

There are many factors that must be considered when building a mass spectrometry laboratory. While some are listed below, others include, but are not limited to return on investment, payment plans offered by each vendor (capital purchase vs. reagent rental vs. paying over several years), available space at your institution, and the versatility of assays that may be run on a single instrument. For a more comprehensive review of how to justify bringing mass spectrometry in-house (70,71).

What analyte(s) do you want to measure?

For labs looking for targeted, quantitative results and/or the ability to confirm samples that screen positive, a triple quadrupole mass spectrometer is plenty sensitive and specific and will likely suffice. If the laboratory wants to use a mass spectrometry-based screen (‘qualitative confirmation’), then an Orbitrap or TOF-MS should be considered. Finally, GC, with or without mass spectrometry, is needed.

Another consideration is whether these instruments will be used for other purposes. Different instruments will be required for quantitatively measuring low concentration hormones versus bottom-up proteomics, and this may shape your purchase selection.

How many instruments do you need to purchase?

Mass spectrometers are not meant to have ~100% uptime like fully automated core laboratory instrumentation. Instead, they regularly require cleaning and replacement of consumable parts. In addition, a vendor-provided preventative maintenance (PM) may take 1–2 days. If it is critical to offer testing daily, then at least two instruments will be needed. The turnaround time required for your patient population may dictate how many instruments, and the staffing required, especially if the lab is expected to run 24/7.

Lab growth and the desire to bring more assays in-house should be considered in advance. If all instruments will constantly be in use, then additional instruments may be needed for new assay method development.

How much mass spectrometry experience does your laboratory staff have?

This is an important consideration when deciding both which type of mass spectrometer to purchase and which vendor to use. It is generally easier to teach a novice user how to use an LC-MS/MS system compared to an Orbitrap or TOF-MS. In addition, LC-MS/MS is commonplace in clinical labs and education may be more accessible. For vendor selection, instruments that are more customizable and consequently more complicated may be better for experienced users compared to less modular instrumentation that may be simpler and preferred for novice users. In either case, it is also critical to carefully review the training, if any, that is included with the instrument purchase.

Is it more cost effective to use multiple smaller panels or an all-inclusive larger panel for confirmatory testing?

Smaller, drug class-specific confirmation panels are often easier to develop, minimizing the time the assay spends in production before going live. They also require fewer analytes in the calibrator, QC, and internal standard, saving on reagent costs. However, if a sample reflects screens positive for multiple drug classes, then that single sample will be prepared and analyzed multiple times, requiring more technical staff time (often the most expensive component of assay costs). A larger, all-inclusive confirmation panel minimizes the technical hands-on time because each sample only undergoes sample preparation, but each sample will require more time for data review, if chromatograms are reviewed manually. The reagent cost per sample will also be higher because there will be more analytes in the calibrators, QC, and internal standard. These considerations must be weighed against reimbursement rates, which are paid only once per sample, regardless of how many times it is run for confirmation.

Result interpretation

Toxicology result interpretation may seem simple at first glance, but is complicated by confounding metabolic pathways, impurities, recognition of minor metabolites, misunderstanding the role of presumptive versus definitive testing, and lack of toxicology training. Unfortunately, clinicians frequently do not have the necessary knowledge base to appropriately interpret toxicology results (6,72). A retrospective chart review across five ambulatory clinics revealed that only 55% of urine drug tests had documented interpretation of the toxicology results, and of those, 28% were discordant with the expert laboratory toxicologist interpretation (73). Even within the laboratory, it is estimated that almost one in ten toxicology result interpretations were incorrect on College of American Pathologists (CAP) proficiency testing surveys when performed by laboratory staff, including bench techs and supervisors (74). This highlights the importance of having a trained clinical toxicologist working in the laboratory to help with result interpretation.

The accuracy and ability to detect drug use through traditional screens is often overestimated by clinical staff while confirmation by definitive technology is underestimated. There are common misconceptions about what the immunoassay screens can detect. For example, the opiate screen, designed to detect morphine, will not detect synthetic opioids such as fentanyl, methadone, or tramadol (75). Similarly, a benzodiazepine screen, frequently designed to detect oxazepam, will not detect all benzodiazepines within the class equally, and the high cutoff concentration may miss those who are taking low doses. Conversely, the false positive rate and false negative rate are frequently underestimated. For example, the amphetamine immunoassay screens often have the highest false-positive rate (5,6,40,41,76), but is frequently unconfirmed. The oxycodone immunoassay is designed to detect oxycodone, and not its major metabolites, so a positive result does not confirm ingestion. Confirmatory testing with metabolites should be used instead to ensure that adulteration by spiking the drug into urine did not occur. Finally, the accuracy of confirmatory testing is frequently undervalued, even when interpreted by experts in addiction medicine (77).

Clinical laboratories take a wide variety of approaches to assist in urine toxicology result interpretation. At a minimum, laboratories should consider adding interpretation comments to urine drug screens, highlighting their limitations, and suggesting confirmatory testing when clinically appropriate. Hybrid screen and direct confirmation ordering panels can simplify this process. If a patient is prescribed a drug and is expected to be positive (for example, clonazepam), then it would be best to send directly for confirmation for that test (benzodiazepines) and screen for the remainder of the drug classes. This hybrid approach is commonly employed for pain management patients undergoing compliance monitoring for opioid use. The CAP offers up to date guidelines and practical advice on toxicology testing and reporting (1).

Similar to screens, laboratories should consider adding comments to confirmatory results that indicate from where a particular analyte may arise. For example, “Morphine may result from codeine, heroin use, morphine-containing drugs, or products containing poppy seeds.” Given the complex metabolic patterns for many drugs, particularly opiates and benzodiazepines, it is also useful to provide a list of expected metabolites for each drug to clinicians. An example of this is provided in Table 3. One limitation of this approach is that the relative concentration of drugs can be very telling, which may not be captured. Interpretive sign outs of every result by a toxicology expert is incredibly beneficial (78), but it is often time-consuming and often not practical for high-volume laboratories.

As technology advances, so does our ability to automate every aspect of clinical testing. To aid in result interpretation without requiring time consuming review and sign out, AI may be employed. Recently, one institution developed a web-based application to improve ordering, interpretation, and accurate resulting of drug confirmations (79). With the quickly advancing use of AI, sophisticated programs may be developed to automatically interpret results and answer clinical questions. Caution should be taken when using these tools. While they can save time, the interpretations should still be reviewed by a trained clinical toxicologist for accuracy before widely implementing. If successful, these tools can improve the efficiency and decrease burnout for laboratory staff.

Validation and QC systems

The selection of appropriate methodology in toxicology testing must be accompanied by rigorous validation and ongoing QC to ensure reliable and clinically meaningful results. According to the CAP, an analytical verification is the process by which a laboratory determines that a test performs according to the manufacturer’s specifications (80). If an FDA-cleared assay is modified, or a test is developed by an individual laboratory, then it requires a validation. A validation is the process used to create objective evidence that a laboratory-developed test (LDT) delivers reliable results for the intended purpose. The difference between these is significant, but both provide confidence that a test can consistently measure an analyte at clinically relevant concentrations under defined conditions.

When verifying the performance of an FDA-cleared assay, a CAP-accredited laboratory must assess the accuracy, precision, reportable range, and any other analytical performance characteristics required to determine if the test is appropriate for clinical testing. For an LDT, on the other hand, the lab must establish the analytical accuracy, precision, sensitivity, specificity, reportable range, carryover tolerance, and other relevant performance characteristics and ensure that they are adequate for patient testing. This is a significantly larger lift for a clinical lab, but fortunately guidance is provided by societies like the Clinical & Laboratory Standards Institute (CLSI). Most notably, they have guidance documents for mass spectrometry-based assays (81), which are almost always considered LDTs at this time. For additional information, we suggest consulting the textbook “Practical Guide to Implementing Liquid Chromatography Mass Spectrometry in Clinical Laboratories” (82).

Beyond initial validation, quality assurance (QA) systems are essential for maintaining assay performance over time. Both immunoassay and mass spectrometry-based assays use calibrators, QC material, and external quality assessments, such as proficiency testing, to monitor assay accuracy and precision. Mass spectrometry-based assays, however, require more frequent calibration, often with every batch of testing, and have additional required quality metrics. These include, but are not limited to, an acceptable peak shape, minimum signal of an isotopically labeled internal standard, and the presence of two product ions at an expected ratio (82).

Adherence to standardized validation and QC practices is critical, not only for regulatory compliance, but also for clinical decision-making. Inadequate validation or poor QC can lead to false results that may carry significant medical, legal, or social consequences, particularly in contexts such as pain management, forensic evaluation, and neonatal testing. Ensuring that toxicology methods are both validated and continuously monitored through robust quality systems enhances the credibility of laboratory findings and supports their integration into patient care (1).

Limitations

While this review provides a broad synthesis of specimen types, current technologies, analytical considerations, and interpretive challenges in toxicology testing, certain limitations should be acknowledged. As a narrative rather than systematic review, the selection of included literature was not intended to be exhaustive and may be influenced by publication availability and scope. In addition, the rapid pace of technological advancement—particularly in mass spectrometry platforms and emerging matrices such as breath and fingerprints—means that some developments published after the time of writing may not be fully captured. Previous articles have often focused on narrower aspects of the field, such as correlation between specimen types (12), common causes of false positives in drug screens (5), or knowledge gaps in toxicological interpretation (74,76). Peters and Wissenbach (2023) provided a comprehensive overview of mass spectrometry technologies (65). In contrast, our review integrates these domains to present a more holistic perspective across specimen selection, screening and confirmation strategies, and interpretive challenges. Nonetheless, further systematic reviews and meta-analyses will be valuable to quantitatively assess assay performance and clinical utility across diverse settings.

Conclusions

In clinical toxicology, selecting the appropriate testing methodology and specimen type requires a nuanced understanding of matrix-specific limitations, analytical performance, and clinical context. Immunoassay screens, while rapid and cost-effective, are prone to both false positives and negatives, particularly as drug diversity increases. Definitive methods like LC-MS/MS offer superior sensitivity and specificity, but demand greater technical expertise and longer turnaround times. As technologies evolve toward automation, high-throughput workflows, and AI-driven interpretation, the clinical laboratory must remain adaptable, prioritizing education, expert interpretation, and thoughtful implementation to ensure accurate, meaningful results that inform patient care.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the Narrative Review reporting checklist. Available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-25-23/rc

Peer Review File: Available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-25-23/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-25-23/coif). K.L.J.D. reports receiving professional development funds from the University of Utah and ARUP Laboratories for conferences attendance and travel. J.A.H. reports receiving professional development funds from Harvard Medical School for conferences attendance and travel. The other author has no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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