The continuous growth in the number of drugs of abuse – including designer drugs – significantly impacts the lives of many. Laboratories involved in drug analysis face the daunting challenge of reliably identifying and quantifying drugs and delivering accurate and timely results. Two types of laboratories, clinical research and forensic toxicology laboratories, play different roles. For instance, clinical research toxicology laboratories deal with many queries on a daily basis and may be involved with therapeutic drug monitoring research or toxicology studies associated with the identification and quantitation of illicit drug use. Results obtained from forensic toxicology laboratories, on the other hand, are often used as evidence in legal proceedings, and could also provide evidence (for example) that a newborn was exposed to a substance of abuse.
Despite their differing roles and focus areas, some of the challenges experienced by both types of toxicology laboratories are similar. Both must test for a wide range of parent drugs and metabolites across different biological matrices, receive a large volume of time-sensitive samples, and are expected to deliver accurate results, quickly.
A wide array of analytical technologies, from immunoassays to gas chromatography, have been used in this domain. In the last decade, toxicology laboratories have experienced the benefits of using liquid chromatography (LC) coupled to mass spectrometry (MS), obtaining data with increased selectivity and specificity. However, the protocols associated with these technologies are typically time-consuming and use costly solvents and consumables – resulting in inefficiencies and large sample loads. Together, these drawbacks can limit a laboratory’s ability to meet sample analysis targets and explore new scientific questions.
In recent years, paper spray ionization has garnered interest in clinical research1 and forensic toxicology laboratories2, due to its time-saving workflows and robust quantitative and qualitative MS-based analysis. In this article, we explore the implementation of paper spray technology for developing a streamlined, reliable method for analysis of drugs of abuse across a range of biological matrices.
Pressure to perform: Why clinical research and forensic toxicology laboratories face backlogs
Clinical research and forensic toxicology laboratories are often high-pressure workplaces, due to a number of contributing factors. The analysis of drugs is inherently complex; biological samples processed in the laboratory may include blood, plasma, serum and urine – and each sample type may require a different preparation method. In addition, the analytes themselves vary with novel compounds of interest continuing to arise. Therefore, analysts must ensure that the developed methods are sufficiently robust and reproducible to enable routine identification and quantification of the targeted drug classes across a range of concentrations.
While conventional LC-MS methods offer short run times, the associated sample preparation in some cases can be time-consuming, significantly increasing the time to results. In such situations, the sample must first be prepared for injection into the LC column through a series of extraction steps that remove undesired analytes and background matrix interferences. In addition to potential time-consuming sample preparation processes, maintenance steps are needed to ensure that the column is thoroughly cleaned between samples and users, which involves further generation of environmentally toxic solvent waste.
As discussed above, owing to the constantly increasing sample load, clinical research and forensic toxicology laboratories in today’s world cannot afford time-consuming LC-MS methods, unexpected equipment downtime and sluggish data processing software. In addition, the manual tasks, where and when needed, can potentially be the source of significant errors. While MS can offer significant advantages, some laboratories involved in drug analysis consequently use immunoassays for their routine testing. Given their limitations in dynamic range, high cost, challenges with selectivity and specificity, and potential for antibody cross-reactivity, immunoassays are, however, not ideally suited for clinical research and forensic toxicology analysis laboratories. Inefficiencies and costs associated with conventional immunoassays increase the cost per sample, and often lead to a backlog of time-critical samples.
Transforming drug analysis with paper spray ionization
Since the pioneering work involving ambient MS in the mid-2000s, paper spray technology has become a popular ambient ionization technique.3, 4 5 When coupled to triple quadrupole MS, paper spray technology can enable a wide range of applications. The robustness, reliability, speed, sensitivity and simplicity demonstrated by this combined technology can enable several clinical research application areas such as, drugs of abuse and therapeutic drug monitoring, etc. Studies have shown that detection limits of low ng/mL and below are obtainable directly from blood, which is sufficient for the detection of most suspect drug candidates. With a total analysis time of less than two minutes, paper spray ionization combined with triple quadrupole MS is becoming popular for its time-saving method and ability to generate high quality data.
One of the most appealing aspects of paper spray technology coupled to MS is that no sample preparation is required. Rather than working through time-consuming manual extraction steps, liquid samples are simply spotted directly onto paper and allowed to dry.5 Following the addition of a solvent to facilitate transport and ionization, a high voltage is applied to the paper via a probe, and the sample is extracted and ionized. The resulting electrospray-like event at the tip of the paper enables a chromatogram of the ion current to be collected, usually for one minute or less, for mass spectrometry analysis.
Within the many fields and applications of clinical research and forensic analysis, rapid and streamlined analytical technologies are highly sought after. Paper spray technologies have found their place across a range of industries, including in the detection of illicit drugs. The simultaneous quantitation of eight drugs of abuse in whole blood was a milestone that clearly demonstrated the value of paper spray ionization; drugs including amphetamine, methamphetamine, morphine and cocaine were quantified from a single blood spot.6 With less than 10 µL of sample, limits of detection for all drugs were below typical physiological and toxicological levels. What’s more, the technique also lends itself to potential use outside of clinical research and forensic laboratories in point-of-care applications, such as in road-side drug testing and employment and workplace testing.6,7
Within clinical research laboratories, paper spray technology is set to transform a range of application areas. In drug monitoring research, for example, paper spray technology enables clinicians to select individualized drug doses rapidly and accurately – a feature that is aspired to but is difficult to achieve with most conventional analytical methods. Enabling an accurate therapeutic research regime can be hugely beneficial and has been explored as an option for those requiring antifungal medication. Invasive fungal infections are highly prevalent in pediatric populations with compromised immune defenses, such as those undergoing chemotherapy or recovering from organ or bone marrow transplants. In a recent study, simultaneous quantitation of five triazole antifungal agents was achieved by paper spray-MS8. No sample preparation was required, and all detection limits were well below clinically relevant concentrations – an important step towards enabling frequent therapeutic drug monitoring research.
The rise of paper spray technology, with its direct-analysis method, also allows room for integration of automation into workflows related to analysis of drugs of abuse. Automated plate loaders, barcode-reading capabilities and robotic spotters can be used to reduce benchwork further and enhance reproducibility. Without the need to purify and concentrate samples for analysis, or spend time and resources on extensive instrument maintenance, paper spray technologies enable laboratories to:
· Increase throughput; further enhanced with automation
· Become more environmentally friendly; minimal waste is generated and very little solvent is used
· Easily access MS analysis through a single instrument
· Achieve high quality, robust, sensitive MS and MS/MS data easily regardless of user expertise or matrix complexity
· Minimize maintenance; robust mass spectrometers can run hundreds of samples unattended without requiring frequent cleaning
Another strength of paper spray ionization lies in its flexibility – not only in the analytes that can be quantified, but also the range of matrices and disciplines to which the technique can be applied. In clinical research and forensic toxicology analysis, there is often a need to examine samples other than whole blood. As such, the technique is currently being explored for alternative matrices, such as the detection of drug metabolites in sweat and urine,2 and the screening and quantification of drugs of abuse in dried urine spots across a range of compound classes.9
How FAIMS technology further enhances drug analysis
With the absence of sample preparation steps in paper spray technology, background interference from the paper substrate has been identified as a potential limiting factor for the accurate quantitation of compounds at particularly low concentrations.10 However, field asymmetric ion mobility spectrometry (FAIMS) has emerged as a promising strategy for increasing the signal-to-noise ratio of MS signals. Essentially, FAIMS filters potentially interfering compounds by providing an additional dimension of separation based on differential ion mobility and involves the application of an asymmetric waveform between a set of electrodes. By alternating between strong and weak electric field strengths, ion mobility is impacted, and target ions pass through the separation region, while non-target analytes are neutralized on the electrode walls.
By integrating FAIMS technology with paper spray ionization, clinical research and forensic toxicology laboratories may expand their analytical capabilities and offer more analytical tests. Several studies suggest that FAIMS is an ideal tool for enhancing direct analysis methods, including the effective separation of morphine, hydromorphone and norcodeine – key drugs and metabolites commonly encountered in forensic toxicology.11 The integration of FAIMS with paper spray technology is an effective strategy for reducing limits of quantitation, enabling laboratories to quantify drugs at even lower concentrations without increasing analysis time.11
Embracing efficient and direct mass spectrometry-based drug analyses
Forensic and clinical research laboratories play a major role in research decisions and forensic investigations and are under constant pressure to analyze time-sensitive samples. Consequently, there is a need for analytical techniques that can support quick turnaround times, while ensuring accuracy, robustness of the method and reproducibility of results.
Recently, paper spray ionization has garnered interest in the fields of forensic toxicology and clinical research for its ability to remove barriers to efficiency. Paper spray technologies remove the need for sample preparation – a major contrast to the time-consuming extraction steps required for conventional chromatography-based MS methods. The addition of FAIMS technology further enhances analysis, by increasing selectivity and reducing limits of quantitation. As has been established across a range of applications, paper spray ionization is the ideal technique for forensic toxicology and clinical research laboratories as it enables the rapid, low-cost quantitation of analytes across multiple matrices – a powerful recipe for removing the relentless backlog of time-critical samples.
1. Chiang S, Zhang W, Ouyang Z. Paper spray ionization mass spectrometry: recent advances and clinical applications. Expert Review of Proteomics. 2018;15(10):781-789. doi:10.1080/14789450.2018.1525295
2. Linhares ALF de A, Yonamine M. Analysis of biofluids by paper spray-MS in forensic toxicology. Bioanalysis. 2020;12(15):1087-1102. doi:10.4155/bio-2020-0160
3. Takáts Z, Wiseman JM, Cooks RG. Ambient mass spectrometry using desorption electrospray ionization (DESI): instrumentation, mechanisms and applications in forensics, chemistry, and biology. Journal of Mass Spectrometry. 2005;40(10):1261-1275. doi:10.1002/jms.922
4. Cody RB, Laramée JA, Durst HD. Versatile New Ion Source for the Analysis of Materials in Open Air under Ambient Conditions. ACS Publications. 2005;77(8):2297-2302. doi: 10.1021/ac050162j
5. McBride EM, Mach PM, Dhummakupt ES, et al. Paper spray ionization: Applications and perspectives. TrAC Trends in Analytical Chemistry. 2019;118:722-730. doi:10.1016/j.trac.2019.06.028
6. Espy RD, Teunissen SF, Manicke NE, et al. Paper Spray and Extraction Spray Mass Spectrometry for the Direct and Simultaneous Quantification of Eight Drugs of Abuse in Whole Blood. Analytical Chemistry. 2014;86(15):7712-7718. doi:10.1021/ac5016408
7. Snyder DT, Pulliam CJ, Ouyang Z, Cooks RG. Miniature and Fieldable Mass Spectrometers: Recent Advances. Analytical Chemistry. 2015;88(1):2-29. doi:10.1021/acs.analchem.5b03070
8. Skaggs CL, Ren GJ, Elgierari ETM, et al. Simultaneous quantitation of five triazole anti-fungal agents by paper spray-mass spectrometry. Clinical Chemistry and Laboratory Medicine (CCLM). 2020;58(5):836-846. doi:10.1515/cclm-2019-0895
9. Michely JA, Meyer MR, Maurer HH. Paper Spray Ionization Coupled to High Resolution Tandem Mass Spectrometry for Comprehensive Urine Drug Testing in Comparison to Liquid Chromatography-Coupled Techniques after Urine Precipitation or Dried Urine Spot Workup. ACS Publications 2017;89(21):11779-11786. doi: 10.1021/acs.analchem.7b03398
10. Tsai C-W, Tipple CA, Yost RA. Integration of paper spray ionization high-field asymmetric waveform ion mobility spectrometry for forensic applications. Rapid Communications in Mass Spectrometry. 2018;32(7):552-560. doi: 10.1002/rcm.8068
11. Manicke NE, Belford M. Separation of Opiate Isomers Using Electrospray Ionization and Paper Spray Coupled to High-Field Asymmetric Waveform Ion Mobility Spectrometry. Journal of The American Society for Mass Spectrometry. 2015;26(5):701-705. doi:10.1007/s13361-015-1096-z
For research purposes only. Not for use in diagnostic procedures.