All substances have the potential to produce harmful effects in the human body, depending on the level of exposure. Determining the identity of substances and the level of exposure plays a key role in mitigating their toxicological effects. In order to handle a variety of substances, from trace metal ions to small organic molecules such as therapeutic drugs and drugs of abuse, toxicological testing laboratories therefore require techniques capable of detecting, identifying, and quantifying analytes of interest in biological samples such as blood, urine, plasma, and other bodily fluids. These methods must be analytically sensitive and selective, and they must offer fast turnaround times and ease of use.
The limitations of immunoassays
Historically, immunoassays have been used for drugs-of-abuse screening and therapeutic drug monitoring in patients. While early assay methods employed radioactive isotopic labels, today many immunoassays are based on enzymatic, fluorescence, or chemiluminescence detection techniques.
Immunoassays are typically used at the point of care, and are based on visual readouts such as color changes and the appearance of diagnostic markers. While these approaches are highly sensitive, can be relatively easy to use, and require little sample preparation, their main limitation is analyte specificity. Because of structural similarities between a parent drug and its metabolites, immunoassays cannot always distinguish among similar compounds of interest. Additionally, immunoassay methods require specific antibodies to identify target analytes, meaning that developing new tests is often time- and resource-intensive.
The rise of chromatography-based methods
As a result of the limitations of immunoassays, approaches based on mass spectroscopy (MS) detection coupled with chromatographic separation have steadily become more common for drug screening and monitoring applications in recent years.
Early use of MS for drugs-of-abuse assessment was largely combined with gas chromatography (GC) separation approaches. However, while GC-MS approaches offer high resolution separation for a range of drug molecules of interest, the technique is limited to the analysis of low molecular weight analytes that are sufficiently stable and volatile at temperatures below 300 degrees. For polar or thermally unstable analytes, time- and labor-intensive chemical derivatization is required, which can also involve sample cleanup and separation steps, prior to GC-MS analysis.
These limitations mean that GC-MS is not amenable to many larger, clinically relevant analytes of interest. Liquid chromatography, on the other hand, is well suited to handle a much broader range of analytes.1 With its high levels of sensitivity and selectivity, the use of LC-MS/MS-based techniques for applications such as drugs-of-abuse analysis and therapeutic drug monitoring has rapidly increased, and LC-MS/MS is becoming a powerful tool for the routine analysis of a wide range of biological samples.
Sample preparation challenges
One of the most significant factors currently limiting the effectiveness of LC-MS/MS for clinical use is the complexity of biological samples. The direct addition of samples to the instrument—the so-called “dilute and shoot” approach—is predominantly used for less complex biological matrices such as urine. For more complex specimens such as blood or plasma, matrix components can affect LC-MS ionization through ion suppression/enhancement or matrix effects.
However, various sample preparation techniques are available to improve the quality of LC-MS data. Extraction and cleanup techniques such as protein precipitation (PP), liquid-liquid extraction (LLE), and solid phase extraction (SPE) are well suited for a range of matrices such as blood plasma and other bodily fluids.1 One of the most commonly used methods, LLE, typically employs nonpolar solvents and can result in poor recovery of phospholipids and polar compounds. SPE can be a more effective option for sample cleanup and offers high recovery rates, reproducibility, and suitability for a wide range of analytes and sample matrices.
Turbulent flow is an alternative and cost-effective on-line extraction method that offers high-throughput sample cleanup for the analysis of small molecules in complex matrices such as biological samples.2 The technology is based on a combination of size-exclusion and traditional column chemistries, designed to separate macromolecules such as proteins from smaller analytes based on differential mass transfer effects. The columns used for this application are packed with large, irregularly-shaped particles of various stationary phase chemistries, which separate the analytes based on rate of diffusion of the particular chemistry.
Drugs-of-abuse screening is one of the most important applications in toxicological analysis, due to the serious health-related and societal harm associated with the abuse, misuse, and diversion of these substances. Typically, screening approaches are based on an initial immunoassay panel, followed by confirmation by MS-based techniques.
The most commonly used LC-MS/MS strategies for routine drug testing panels are based on multiple reaction monitoring approaches, employing triple quadrupole mass spectrometers. The technique is based on the use of electrospray ionization methods, followed by analyte isolation and fragmentation through a three-stage quadrupole instrument. Here, the first quadrupole isolates a molecular or “precursor” ion of interest, the second acts as a collision cell to fragment this precursor ion, and the third is used as a mass analyzer to detect the daughter ions. Since the technique identifies not only the molecular ion but also the fragment ions and the LC retention time, LC-MS/MS is an effective and highly selective approach for the confident identification and quantitation of large panels of target analytes.
In recent years, advances in the sensitivity and mass accuracy of high resolution MS technology have led to the development of fast and effective untargeted approaches for the detection and quantitation of drug molecules. Given the challenges posed by the global rise in the number of novel psychoactive substances entering the market, these full-scan approaches are proving to be particularly useful, not only because of the need to screen for a much broader range of analytes, but also for the rapid identification of unknown compounds of interest.
Therapeutic drug monitoring
Therapeutic drug monitoring is used to guide the selection of treatment regimens, assess compliance, and monitor efficacy and toxicity.3 This is particularly important for drugs with a narrow therapeutic window or those where pharmacodynamics is highly variable among individuals.
For treatments based on immunosuppressant drugs, for instance, constant monitoring is necessary to establish an appropriate therapeutic dose. Under-dosing of immunosuppressants in patients undergoing tissue transplant can result in graft rejection, while overdosing can increase the risk of infection. While a number of immunoassays exist for the monitoring of immunosuppressant drugs, LC-MS/MS is now widely used due to the low consumable cost per test, the high specificity of analysis, and its ability to analyze multiple immunosuppressants using a single test.
For patients undergoing pain management therapies, monitoring is necessary to assess compliance with treatment regimens, due to the potential for abuse, misuse, or drug diversion. Here, urine sampling can be a fast and effective way to screen for panels of drugs and their metabolites to determine whether the patient is complying with the therapy.
There is also an increased interest in drug monitoring for psychiatric medication regimens, especially where adverse effects can be correlated with the concentration of the drug in the blood. For tricyclic antidepressant drugs, for example, such as amitriptyline and desipramine, there is a well-defined relationship between blood concentration and cardiac arrhythmia, and therapeutic drug monitoring is necessary to ensure patient safety.3 With high rates of poor adherence to treatment regimens among psychiatric patients, there is also a growing interest in the use of drug monitoring to support compliance.
Trace elemental analysis
The detection of trace elements in the body is another important application in toxicological assessment. The excess or deficiency of certain elements in the body can present a significant risk to health. For example, blood concentrations of lead above 25 μg/dL can lead to physiological problems,3 while low levels of the essential element zinc have been linked to impaired immune system functionality.4
Quadrupole inductively coupled plasma-mass spectrometry (ICP-MS) has emerged as a well-established approach for trace elemental analysis.5 The technique is capable of routine quantitation at detection limits on the parts-per-trillion level for a broad range of elements, and is amenable to a wide variety of biological matrices. ICP-MS is based on a “hard” ionization approach, as the analytes of interest are largely single-atom ions rather than molecular species. Generally, sample analysis is based on a dilute-and-shoot approach; however, cell-based samples typically require additional processing, such as digestion by nitric acid.3
While routine quantitation is typically based on comparison with an external calibration curve, isotope dilution methods involving the measurement of isotope abundance ratios offer enhanced accuracy for quantifying levels of trace elements, as this approach is significantly less prone to matrix effects.5 In addition to routine quantitation, the application of isotopic fingerprinting techniques, such as dilution and ratio measurements, can be used to help identify the source of heavy metal exposure in patients.
Overcoming ICP-MS interference challenges
Despite ICP-MS being a relatively robust analytical method, interference from isobaric molecular ions can be a significant challenge for ICP-MS approaches, and generally results from the recombination of compounds containing elements such as Ar, H, O or CL.3 Improvements in MS resolution are helping to overcome these challenges, with instruments possessing a resolving power greater than 10,000 generally able to overcome most interference issues.
An alternative approach to reduce the impact of interference is the use of a low-pressure reaction cell. Using a reactive gas such as hydrogen or ammonia, the charged ions responsible for interference can be neutralized via a charge transfer reaction, rendering the species undetectable.
While improvements in the analytical sensitivity and mass resolution of MS instruments continue apace, there is also an intense focus on developing more efficient sample preparation strategies and the ability to study a wider range of biological samples. The development of drug screening and monitoring approaches based on the use of exhaled breath, for example, could offer a less invasive alternative to the collection of biological specimens where blood or urine collection is challenging. The approach is already being investigated for drugs-of-abuse screening and therapeutic drug monitoring.6 In addition, use of alternate matrices such as hair and oral fluid are gaining popularity in the clinical lab.
Furthermore, the development of more portable MS devices is creating opportunities for point-of-care applications. Here, the ability to more conveniently monitor therapeutic drug levels in real time could help to improve patient care where there is a need to make dose adjustments quickly, such as during surgery.
Another trend that is set to further enhance productivity in toxicological analysis is the ongoing move toward automation. Across a range of clinical toxicology applications, there is a demand for “push-button” solutions that can be operated by non-specialists. The adoption of increasingly automated techniques is supporting analysts to work more efficiently and return results faster.
Highly sensitive, specific, and applicable to a very broad range of analytes, MS techniques have emerged as the dominant technology for toxicological analysis. The latest advances in MS instrumentation are driving further improvements in the accuracy, reliability, and efficiency of toxicological testing protocols. Coupled with faster and more effective sample preparation steps, MS methods are helping clinicians obtain more information from a wide range of biological specimens to ensure that patients receive personalized treatment tailored to their individual circumstances.
- Viette V, Hochstrasser D, Fathi M. LC-MS (/MS) in clinical toxicology screening methods. Chimia. 2012;6(5):339-342.
- Couchman L. Turbulent flow chromatography in bioanalysis: a review.
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- Rockwood AL, Johnson-Davis KL. Mass spectrometry for clinical toxicology:
therapeutic drug management and trace elemental analysis. Clin Lab Med. 2011;31(3):407-428.
- Haase H, Rink L. Multiple impacts of zinc on immune function. Metallomics. 2014;6(7):1175-1180.
- Rodushkin I, Engström E, Baxter DC. Isotopic analyses by ICP-MS in clinical
samples. Analy Bioanal Chem. 2013;405(9):2785-2797.
- Beck O, Olin AC, Mirgorodskaya E. Potential of mass spectrometry in developing clinical laboratory biomarkers of nonvolatiles in exhaled breath. Clin Chem. 2016;62(1):84-91.
Suparna Mundodi, PhD, serves as Vertical Marketing Manager, Clinical Toxicology, for Thermo Fisher Scientific.