Ion mobility–mass spectrometry (IM–MS) has become a cornerstone bioanalytical technique over the last two decades. Major advances have included new technology and software that enabled higher resolving power measurements; however, chemistry-based approaches, such as derivatization reactions, have also shown tremendous promise. This article demonstrates our work using IM–MS to detect anabolic steroids and describes how this technology could one day find its way into the anti-doping world.

The world of analytical testing for doping violations in sports and athletic competitions has been dominated by mass spectrometry (MS)-based methods for several decades now, dating back to the development of the University of California, Los Angeles (UCLA) Olympic Analytical Laboratory in 1982. The laboratory was founded in advance of the 1984 Olympic Games in Los Angeles, and it is currently one of only two World Anti-Doping Agency (WADA)–accredited laboratories in the United States (the second being the Sports Medicine Research and Testing Laboratory in Salt Lake City, Utah). There are approximately 30 WADA-accredited laboratories worldwide that oversee the testing of athlete samples, according to the World Anti-Doping Code and the annual Prohibited List.

Many of the methods that are used to enforce the World Anti-Doping Code involve a combination of MS and chromatographic separations (gas or liquid chromatography). The most frequently used MS platform for targeted analysis remains the triple-quadrupole mass spectrometer (QqQ, also notated as TQMS or MS/MS), which has been lauded for decades because of its superior sensitivity when operated with selected reaction monitoring (SRM). More recently, high-resolution platforms have become more routine in analytical laboratories because their ability to determine molecular formulae from accurate mass measurements has been joined by improved quantitative capabilities. Despite widespread implementation of modern MS instrumentation and advanced software for data interpretation, much active research focuses on qualitative and quantitative improvements. One such advance has been the commercialization of ion mobility (IM)–MS instrumentation.

Ion mobility spectrometry (IMS) is a gas-phase separation technique first developed in the 1950s for defense applications, including the detection of chemical warfare agents. IMS technology has also been heavily utilized in airport security checkpoints as a rapid screening tool; in fact, tens of thousands of these standalone IMS devices remain in service by government and military entities across the United States. However, the IM–MS technique opened a door to the larger and more complex analytes required in bioanalysis, with groups such as those of David Clemmer at Indiana University, Herbert Hill at Washington State University, and Michael Bowers at University of California, Santa Barbara developing early home-built systems to measure carbohydrates, peptides, and proteins, among other biomolecules.

IMS shares some similarities with MS in that it uses electric fields to manipulate ions for measurement. Unlike MS, which is performed under a high vacuum to increase the mean free path (that is, avoiding collisions between analyte ions and stray gas molecules), IMS operates at pressures as high as 1 atm. This pressure results in many collisions so that analyte ions are separated by mass and by a combination of size, shape, and charge. Historically, IMS has seen applications ranging from the study of native protein structure (misfolding in neurodegenerative diseases) to the separation of small molecule isomers, which may look identical even with today’s highest-resolution mass spectrometers.

In the last 20 years, almost all major MS vendors have released their own version of IM–MS platforms, dating back to Waters Corporation’s release of the Synapt HDMS in 2006. Furthermore, several other companies have also introduced their own “add-ons” that can easily be coupled to existing MS instruments. Such widespread adoption of IM–MS technology has moved this technology past the “emerging” stage and becoming a cornerstone of bioanalytical laboratories.

The Future of Ion Mobility in Anti-Doping?

Despite the widespread adoption of IM–MS technology, routine implementation of IM–MS instrumentation in regulated analyses, such as those performed in clinical testing or anti-doping laboratories, has not happened yet. The basic science performed in our group is largely focused on the application of IM–MS to steroids and related metabolites, with previous studies highlighting its use for analyzing anabolic steroids, bile acids, and vitamin D metabolites, to name a few. As an example, our group has demonstrated improved quantification of target analytes, such as testosterone in human urine (Figure 1) (1). More recently, we started investigating ways in which we can improve the performance of modern instrumentation for applications, including anti-doping.

Early work goes back almost a decade to the first simple IM–MS analysis of steroid isomers. Given their identical exact mass (and oftentimes very similar MS/MS fragmentation patterns), these were ideal targets for mobility separations. The early studies laid the groundwork and demonstrated the potential for IM–MS by separating a series of endogenous steroid isomers (2). However, challenges emerged, including the resolution of stereoisomers such as the epimers testosterone and epitestosterone. Differentiation could be achieved when measuring these species as dimers because of different stacking structures, but these were only observed at high concentrations and were unlikely to be feasible in realistic samples (2,3).

It was during this time that computational modeling was also starting to be seen as a critical complement to experimental IM–MS studies. By using density functional theory, our collaborators could determine the gas-phase structures (with a high degree of accuracy) that accounted for the empirical results we produced. Such studies allowed us to determine the stacking structure of steroid multimers, as well as conformational differences for clinically relevant 25-hydroxyvitamin D3 isomers (3–5). These complementary studies have become routinely incorporated into IM–MS research.

Despite some of the early promise demonstrated, it was clear that the limited resolving power of many of the early IMS instruments would be restrictive for separating structurally similar species such as stereoisomers. In fact, the last decade has seen a collective push toward advancing higher-resolution IMS hardware and software. As an example, researchers at the Pacific Northwest National Laboratory (PNNL) have developed a new IM platform that has enabled serpentine long-path separations and has even been applied to the steroid subclass of bile acids (6). The technology has since been commercialized by Mobilion Systems.

Aside from hardware and software advances, others in the field have focused on using chemistry to modify molecular structure and augment separation capabilities. The inspiration for the work done today dates back to early studies from the groups of Colin Creaser (then at Nottingham Trent University), David Clemmer at Indiana University, and John McLean at Vanderbilt University. Our group has seized upon this inspiration to apply these strategies to the study of steroids, focusing on modifications of alkene, hydroxyl, and carbonyl chemistry. Our most recent work has borrowed from the lipidomics field and specifically ozonolysis and Paternò-Büchi reactions. With ozonolysis, we exposed steroids to ozone, which resulted in cleavage of the C=C double bonds and an opening of the A-ring in isomers testosterone and epitestosterone (Figure 2). The structural flexibility introduced by this cleavage resulted in unique gas-phase structures that could easily be resolved (when the unre- acted isomers were nearly identical). Again, computational modeling results confirmed our experimental studies and demonstrated the differences in the points of interaction with a sodium ion between the ozonolysis products of testosterone and epitestosterone (7). As a follow-up study, we applied UV radiation in the presence of acetone to perform the Paternò-Büchi reaction, which involves radical addition of a carbonyl across a C=C double bond. This was used to modify a series of endogenous steroid isomers, which resulted in unique fingerprint spectra that allowed differentiation (8).

Although IM–MS has yet to become the gold standard in regulated bioanalysis methods (that is, clinical and anti-doping), the technology has shown tremendous promise. It is clear from the trajectory of current research that advances in hardware, software, and chemistry-based approaches to separations will pave the way for its even more widespread implementation.

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 is an Assistant Professor of Chemistry at the Florida Institute of Technology, in Melbourne, Florida. Direct correspondence to: 

Articles

Ion mobility–mass spectrometry (IM–MS) has become a cornerstone of bioanalytical laboratories. New developments could lead to its widespread adoption for regulated bioanalysis methods such as anti-doping testing for anabolic steroids in athletes.

Enhancing the Performance of Analytical Methods to Combat Doping in Sport

Liquid chromatography–mass spectrometry (LC–MS) with electrospray ionization (ESI) is a widely used bioanalytical technique with both qualitative and quantitative applications. Ions are created by electrically charging a stream of droplets from the LC system, which evaporate and leave ions that are transferred to the mass spectrometer. Ideally, these are only from the analyte, but background ions, such as metals, impurities and coeluted species, can react with analytes producing adducts, such as [M + Na]

, and so forth). Although well known, the extent of adduct ion formation and the implications for quantitative analysis and analyte characterization by tandem MS (MS/MS) are not fully appreciated. We summarize the problem and identify areas that should be considered when developing or using electrospray LC–MS.

Modern liquid chromatography–mass spectrometry (LC–MS) is dependent on atmospheric pressure ionization (API) techniques, such as atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI). ESI is widely used for analyzing polar molecules in biological fluids and enables applications such as proteomics and metabolomics as well as analyses in drug development, toxicology, and doping control. It is compatible with a wide range of LC flow rates and usually produces protonated ([M+H]

) ions that can be dissociated to generate structurally informative fragments using collision-induced dissociation (CID). The combined technique is known as LC–tandem MS (MS/MS) because it uses two mass selection steps, one to select a precursor ion and the other to analyze the fragments following CID. Selectively monitoring fragment ions from a preselected precursor ion provides a sensitive quantitative method known as 

 that is widely used in drug development and legal toxicology applications. Analysis of the fragment ions is used for compound identification, either by comparison with spectral libraries or de novo, preferably using a high-resolution analyzer that generates accurate mass values from which likely elemental compositions can be determined. The process has been automated in a technique known as 

, where candidate precursor ions are determined from one mass analyzer scan, individually selected and fragmented, and the product ions analyzed. This technique is the basis of proteomics. With DDA, the selection window is generally 1 Da wide to increase the specificity, but it was recently realized that if the fragments are sufficiently specific (for example, because of high-resolution mass analysis), wider selection windows can be used. This process has become known as 

 and has several advantages, primarily that a wide range of precursor ions can be monitored continuously and that all compounds are fragmented, generating a reproducible permanent record of the sample. For quantitative DIA, as used in proteomics, data analysis is based on the chromatographic behavior of fragment ions known to be produced by the target compounds.

ESI ions are created by electrically charging a stream of droplets from the LC system, which evaporate and leave ions that are transferred to the mass spectrometer. Ideally, these only come from the analyte, but ions from involatile entities, such as metals and impurities present in the sample or from the instrument, can also react with analyte molecules and produce adducts, such as M + Na

. Although these are well-known artifacts, the full extent and implication of these reactions has only been recently realized. For example, Mahieu and others (1) reported in 2017 that 25,000 LC–MS features could be reduced to 1000 and Broeckling (2) in 2016 noted that much of the signal measured in the spectra of known compounds could not be explained. In fact, Broeckling’s paper contained a spectrum of dimethylsuccinic acid (Figure s2 in that paper) where the [M + H]

 ion is weak, and the ions containing sodium and potassium are more intense. However, the bulk of the intensity is because of the multimers (2M and 3M) combined with potassium and also with other, unknown entities. We encountered similar phenomena while building spectral libraries by infusing standards (3) and developed software to recognize the more common species (4); however, like Broeckling, many species remained unidentified, which can be particularly challenging when several compounds are coeluted, as illustrated in Figure 1.

We subsequently developed an approach called multi-layered analysis (MLA) to focus on unidentified ions (Figure 2), which revealed multimers and adducts with calcium, aluminum, iron, and barium, and potentially magnesium, zinc, and molybdenum in our spectrum and calcium, aluminum, and iron in Broeckling’s. These metals apparently arise from parts-per-billion (ppb)-level trace elements in the solvents used (particularly formic acid) although some, like potassium, are present in biological fluids. Although sodium and aluminum are monoisotopic, the others have patterns that can aid interpretation, such as barium, but they can also overlap and produce more complex patterns (5).

As part of this study, we calculated adduct masses using effective adduct mass (MA) which is equal to 

 is the mass of hydrogen, plus the number of protons required by the observed charge. Therefore, the basis of barium adducts with a valency of 2 is M+ (Ba – 2H) giving singly charged ions with 

 and doubly charged ions at (M + (Ba–2H) + 2H

)/2. Using this approach emphasized two important facts. First, elements with different masses and valency can produce adducts that are close in mass and can be resolved by their isotope patterns (for example, ions from potassium and calcium differ by 0.009 Da). Second, the same element in different oxidation states can also produce overlapping patterns; for example, Fe(II) and Fe(III) are shifted by the mass of hydrogen, which is 1.007825. Furthermore, adducts can involve combinations of elements (for example, M + Na + Al) and the addition of background and coeluted species, making complete interpretation difficult. We also note that sample preparation can also produce byproducts that complicate the analysis, but these are different compounds that may be separated, whereas adducts are ion source phenomena and have the same retention time as the analyte.

Regardless of the exact source and composition of adducts, their presence has important implications for LC–MS analyses, especially for the complex samples often encountered in bioanalysis.

The formation of adducts and ions from reactions between analytes and background ions or between coeluted analytes is highly dependent on the sample matrix, the purity of the reagents and solvents, the cleanliness of the system, and the separation used. Thus, the type and extent of these species is liable to inter-instrument, inter-laboratory, and temporal variation, affecting the intensity of the observed ions, which would impact all of the areas discussed below and may confound long-term studies such as proteomics and metabolomics. The ability to transfer methods between laboratories will also be affected (for example, if the reagents used are from different sources). We speculate that this problem may be part of the reason for the anecdotal difficulty in transferring regulated clinical trial assays between pharmaceutical companies and contract research organizations (CROs).

As noted, adducts can dramatically increase the number of LC–MS features observed, which complicates interpretation of the results. We have noted that this complication is especially true in analyzing high-molecular-weight compounds, such as peptides and glycopeptides, because these have extended 

C isotope patterns and often form multiply charged ions that reduce 

 isotope spacing. As a result, adducts can produce complex overlapping isotope patterns that result from simple mass shifts for adducts that have no isotopes (sodium, aluminum, cobalt, and manganese) or altered patterns if the adduct itself has isotopes or different oxidation states (iron, molybdenum, and cadmium). In some cases, real modifications may be confused with adducts of similar mass, and if these are coeluted, the observed mass may not correspond to either. For example, the effective adduct mass of cobalt is 56.91754, and an acetamide derivative differs by 57.021464, and although the 

 difference of 0.104 is usually resolved, it decreases with increasing charge and the species may eventually merge, giving a peak at 56.96950 (assuming equal intensity), which differs by 0.053 from both species.

Because adducts arise from real analyte molecules, they may reduce the intensity of the expected ions and cause inaccuracy, which could be particularly dangerous in targeted assays, such as SRM and multiple reaction monitoring (MRM), because the full spectrum is not usually recorded and the adduct ions may be undetected. Furthermore, the introduced variability will cause the error to change over time. We also speculate that the lengthy development required for many regulated pharmaceutical assays may, in part, arise from the unrecognized need to control adduct formation to obtain the necessary linear dynamic range.

DDA is a fully automated process controlled by the instrument manufacturer’s software and, to our knowledge, does not consider adduct formation. Thus, adduct ions are as likely to be selected, especially if they are intense or if deep selection is used, resulting in low-quality spectra that are difficult to interpret because adduct ions often fragment poorly. We further speculate that this may be part of the reason that many spectra obtained in proteomic analyses are unassigned. However, we reported that alternative fragmentation techniques can produce spectra from adduct ions with complementary information that aids interpretation (6).

In DIA techniques, such as sequential widowed acquisition of all theoretical fragment ion mass spectra (SWATH) (7), all ions are fragmented and adduct ions may be useful. For example, in a simple metabolomics study (8), we noted a change in an LC–MS feature that was identified as a potassium adduct and, because this was SWATH data, we followed the fragmentation chain and identified the glucuronide derivative of a known metabolite. Further, SWATH allows comparison of MS

Although well known, the extent and implication of adduct ion formation may not have been fully appreciated. As discussed, adducts often cause additional complexity, may reduce sensitivity in targeted analyses, and introduce uncontrolled variability that challenges reproducibility. However, they may also provide opportunities for alternate fragmentation methods that could be exploited by instrument manufacturers. They can also help confirm the molecular weight, especially if the [M+H]

 ions are absent and may reveal structural information if formation is dependent on the structure.

Regardless, because moieties potentially causing adducts are unavoidable in biofluids and reagents, it is important that the possibility of adduct formation (especially with unusual or unexpected elements) is considered and treated appropriately or that precautions are taken to avoid undesirable consequences.

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 runs Ron Bonner Consulting in Newmarket, Ontario, Canada. 

 is with the Department of Inorganic and Analytical Chemistry at the University of Geneva, in Geneva, Switzerland. Direct correspondence to: 

Although well known, the extent of adduct ion formation in LC–ESI-MS/MS, and the implications for quantitative analysis and analyte characterization are not fully appreciated. We explore this problem and explain the implications for reproducibility, quantitative and qualitative analyses, and DDA and DIA.

The Origin and Implications of Artifact Ions in Bioanalytical LC–MS

Benchtop liquid chromatography (LC)–mass spectrometry (LC–MS) and benchtop gas chromatography–MS (GC–MS) have been the gold standard in bioanalysis for decades, employed in clinical, forensic, and anti-doping laboratories worldwide. However, traditional instrumentation is subject to limitations that severely restrict the speed at which samples can be analyzed, the ease with which materials can be prepared, and the location where the analysis must be performed. With the advent of ambient ionization and portable mass spectrometers, the ability to perform rapid, on-site analysis is fast becoming a reality. Without the need for extensive sample preparation, analysis times as low as several seconds, and the ability to perform analysis remote from the laboratory, deployable mass spectrometers have the potential to revolutionize bioanalysis. However, several challenges must be tackled before this innovative technology can be widely adopted.

In the field of biochemical analysis, liquid chromatography–mass spectrometry (LC–MS) and gas chromatography–MS (GC–MS) have become essential tools for identifying and quantifying analytes in biological matrices. They are widely used in clinical laboratories for diagnostics, in anti-doping for detecting controlled performance-enhancing substances, and in forensics for detecting illicit substances, to name a few application areas. Despite the widespread use of these crucial techniques, there are several key limitations of traditional MS approaches that warrant the development of alternative technologies.

Biological samples, such as blood and urine, are highly complex, containing an abundance of interfering analytes. As such, raw samples are not amenable to analysis by LC–MS or GC–MS, necessitating sample cleanup, extraction, and, in some cases, chemical derivatization. Such sample preparation steps can be laborious, are highly invasive to the sample, and can be costly. Traditional techniques employ chromatography for the separation of sample components. Although important for the accurate identification and quantification of compounds, the chromatographic separation of analytes is a time-consuming process, with some run times being more than an hour per sample. In addition, these techniques can be costly and necessitate the use of expensive gases and solvents and, in the case of LC, environmentally damaging through the substantial quantities of solvent waste. Given the relatively large footprint of some instruments and the need for gas supplies and vacuum pumps, traditional mass spectrometers are by no means portable. As a result, samples must be transported to the laboratory for analysis, inevitably taking time and often necessitating specific storage and transportation requirements, such as low temperatures to preserve the biological materials.

Given the growing need for high-throughput and even portable analysis, recent developments in analytical technology have the potential to revolutionize bioanalysis both in the laboratory and in the field.

Ambient Ionization and Portable Mass Analyzers in Bioanalysis

Over the past two decades, an astonishing amount of research has focused on pushing analytical techniques from the laboratory into the field (1). Research has targeted both the development of novel ion sources and miniaturized mass analyzers suitable for on-site analysis. Ambient ionization mass spectrometry (AIMS) techniques utilize ion sources capable of functioning in the ambient air, eliminating the need for hefty pumps to maintain vacuum. AIMS allows for the rapid, direct analysis of solids, liquids, and gases without lengthy sample-preparation procedures or time-consuming chromatographic separations. When combined with portable mass spectrometers, miniaturized analyzers are capable of being transported in the back of a vehicle or even as a backpack, which is shown in Figure 1 (2). Combining AIMS with portable mass spectrometers creates and forms a powerful new toolkit that has the potential to change the way we can deploy analytical testing across countless fields of study.

The potential utility of AIMS in the clinical area (that is, to perform rapid diagnostics of different diseases or to achieve the near-real-time measurement of medicines in the body) was identified in the early days of AIMS development. Despite the growing number of AIMS techniques available, a few key techniques have been demonstrated to be particularly useful in clinical bioanalysis. For example, paper spray ionization (PSI) coupled to MS (PSI-MS) is an ambient ionization technique that involves the application of a sample to a triangular piece of paper in front of the mass spectrometer inlet. The subsequent application of a high voltage induces near-instantaneous ionization and detection of analytes. Developed by the Cooks group in 2010 (3), early applications of PSI focused on therapeutic drug monitoring, which demonstrated the ability to rapidly detect a range of medicinal drugs in dried blood (4,5). The technique has since been highlighted as a potential tool in biomarker discovery and disease diagnostics, suitable for the analysis of both body fluids and tissue samples (6,7). Although these studies coupled PSI with benchtop mass spectrometers, other studies have successfully utilized PSI with portable instrumentation for analyzing drugs as bulk materials (8–10) and in dried blood spots and urine (5,10,11). As an alternative electrospray (ES)-based AIMS technique, probe ES ionization (PESI) incorporates a solid needle that is simply dipped into a sample and positioned in front of the mass spectrometer inlet. Then, a high voltage is applied to induce ionization and instantaneous analyte detection. Developed by Hiraoka and others in the mid-2000s (12), PESI was found to be a versatile technique for the direct analysis of raw biological fluids (13–15) and body tissues, particularly for the differentiation of cancerous and healthy tissue samples (16,17).

More recently, the potential use of AIMS for real-time analysis in anti-doping has been identified. In sports, the use of performance-enhancing substances by athletes is strictly controlled by the World Anti-Doping Agency (WADA). Analytical techniques such as LC–MS are employed for the detection of prohibited substances and their metabolites in biological samples, particularly urine and blood. Given the number of athletes tested during major sporting events, deployable and rapid analytical testing would be of substantial benefit to the field. Some early efforts have been made to apply such techniques in this area, with the atmospheric pressure solids analysis probe (ASAP) being a prime candidate as the technique of choice in anti-doping. ASAP-MS incorporates a sampling probe that is introduced to the sample matrix and then exposed directly to the plasma of an atmospheric pressure chemical ionization (APCI) source (18). The technique was most recently demonstrated for the rapid (1 min) analysis of anabolic steroid esters with automated data processing using a commercially available compact ASAP-MS system (19). This system incorporates the efficient functionality of ASAP, a compact transportable mass analyzer, and a simple real-time sample classification system, all suitable for use by a non-expert. Importantly, this particular instrument (Waters Radian ASAP-MS) can be purchased with an built-in diaphragm pump, meaning that the entire system is available within a single unit with only the need for an external nitrogen gas source. Although this model offers reduced sensitivity over a rotary pump alternative, the all-in-one approach offers a distinct advantage when considering “deployability” of an instrument. Despite the clear potential of AIMS in anti-doping, a select few other techniques have been applied in this area. Rossini and others recently used PSI–MS to detect anabolic agents and diuretics in raw urine, demonstrating the rapid detection of pertinent analytes at significantly lower levels than the WADA required limits of detection (20). Similarly, Görgens and others applied the same technique in analyzing WADA-prohibited substances in raw urine, with a particular focus on the detection of polar compounds, which can be challenging to analyze even when using traditional LC–MS techniques (21). Direct analysis in real time (DART), one of the first ambient ionization techniques to be described, has furthermore been demonstrated in analyzing products and raw urine containing dimethylamylamine, a WADA-controlled stimulant (22). Finally, coated blade spray (CBS), a technique that utilizes a coated stainless steel blade for analyte enrichment and ionization (see Figure 2), has been utilized in detecting controlled substances in biofluids (23,24). Furthermore, CBS ion sources have been demonstrated to be a suitable tool for deployment, with a recent study developing a universal serial bus (USB)-powered CBS source with a transportable MS for on-site analysis (25).

Traditional analytical techniques, such as LC–MS and GC–MS, are further widely utilized in the forensic science sector, including in the analysis of biological materials. Biological samples, such as blood and urine, may be subject to analysis for detecting the presence of medicinal or illicit substances in the bloodstream of a perpetrator or victim and, more recently, in efforts to develop techniques for the identification of forensically relevant biofluids. AIMS techniques have already been widely applied across forensic bioanalysis, demonstrating the potential to analyze biological materials rapidly both in the laboratory and on-site at incident scenes.

Multiple techniques have been utilized to rapidly detect illicit drugs (27–29) and toxins (30) in body fluids. AIMS techniques have furthermore been utilized for the classification of forensically relevant biological fluids (15,31–33), although this application is still in its infancy and not conducted outside of the realms of research. Given the nature of criminal investigations, the need for rapid sample analysis is crucial for the timely investigation of criminal matters, and the ability to perform on-site analysis of biological materials at an incident scene could vastly improve the efficiency of investigations.

As with all new technological advances, ambient ionization sources and portable mass analyzers are not without their problems, and further development is needed before such techniques can be truly deployed. Operating in the atmospheric environment, AIMS techniques must often function with noisy backgrounds that can introduce a substantial amount of interfering compounds, resulting in ion suppression, affecting levels of sensitivity along with reproducibility and accuracy of quantification. Moreover, the need for clean nitrogen air sources decreases the overall “deployability” of the equipment; therefore, further work into the potential for non-specialist gases to be used could provide game-changing opportunities to the portable MS space. Research is increasingly focusing on the improvement of AIMS performance to tackle these problems, but current technology does not yet quite compete with more established technologies. The majority of AIMS techniques developed to date utilize homemade ion sources that may not be practical for widespread use by clinical and government laboratories. In recent years, some ion sources have been successfully commercialized and others will no doubt follow, given the increased interest in such technologies across multiple fields of work. Such techniques also necessitate highly specialized knowledge for operation, whereas widespread deployment of portable technologies would ideally be usable by non-experts. In recent years, there has been a trend toward the development of simple “point-and-shoot” systems with automated data analysis to provide the user with simple real-time results (34,35).

As for deployable mass analyzers, portable and transportable mass spectrometers are not new technology, but their performance cannot currently compete with the high-resolution benchtop instruments widely employed in many laboratories. However, an increasing number of instrument manufacturers have introduced transportable systems that can be readily deployed for on-site analysis remote from the laboratory. Truly portable systems, such as handheld or backpack-style systems, have been developed (36), but they are not yet widely available. Extensive research is ongoing to tackle the existing limitations of ambient ionization sources and portable instrumentation, the results of which will no doubt lead to major changes in bioanalysis and many other fields of analytical sciences.

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(3) H. Wang, J. Liu, R.G. Cooks, and Z. Ouyang, 

(4) N.E. Manicke, P. Abu-Rabie, N. Spooner, Z. Ouyang, and R.G. Cooks, 

(5) Z. Zhang, W. Xu, N.E. Manicke, R.G. Cooks, and Z. Ouyang, 

(12) K. Hiraoka, K. Nishidate, K. Mori, D. Asakawa, and S. Suzuki, 

(14) K. Yoshimura, L.C. Chen, D. Asakawa, K. Hiraoka, and S. Takeda, 

(15) S. Rankin-Turner, S. Ninomiya, J.C. Reynolds, and K. Hiraoka, 

(18) C.N. McEwen, R.G. McKay, and B.S. Larsen, 

(19) A. Arrizabalaga-Larrañaga, P.W. Zoontjes, J.J.P. Lasaroms, M.W.F. Nielen, and M.H. Blokland, 

(6), 1212–1222 (2020). DOI: 10.1021/jasms.0c00063

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(24) G.A. Gómez-Ríos and J. Pawliszyn, 

(11), 2243–2249 (2020). DOI: 10.1021/jasms.0c00307

(27) S. Saha, M.K. Mandal, and K. Hiraoka, 

(28) G.W. Vandergrift, A.J. Hessels, J. Palaty, E.T. Krogh, and C.G. Gill, 

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, 106610 (2019). DOI: 10.1016/j.vascn.2019.106610

(31) S. Jackson, B.S. Frey, M.N. Bates, D.J. Swiner, and A.K. Badu-Tawiah, 

(32) S. Rankin-Turner, M.A. Turner, P.F. Kelly, R.S.P. King, and J.C. Reynolds, 

(33) S. Rankin-Turner, P.F. Kelly, R.S.P. King, and J.C. Reynolds, 

(34) J.S. Wiley, J.T. Shelley, and R.G. Cooks, 

(39), 13326–13333 (2021). DOI: 10.1021/acs. analchem.1c03087

(36) D.T. Snyder, C.J. Pulliam, Z. Ouyang, and R.G. Cooks, 

 is with the W. Harry Feinstone Department of Molecular Microbiology and Immunology in the Johns Hopkins Bloomberg School of Public Health at Johns Hopkins University, in Baltimore, Maryland. 

 is with the School of Sport, Exercise, and Health Sciences at Loughborough University, in Loughborough, United Kingdom. Direct correspondence to: 

With the advent of ambient ionization and portable mass spectrometers, the ability to perform rapid, on-site analysis is fast becoming a reality. We review the critical recent developments enabling this capability as well as remaining challenges that must be tackled to enable widespread adoption.

Deployable Mass Spectrometry for Rapid On-Site Bioanalysis

Reversed-phase liquid chromatography (RPLC) may have historical popularity, but it is not the only LC technique available to chromatographers. To help balance the choice of chromatographic technique, a discussion of the merits of being flexible when making the choice of LC technique for a given application is presented. Because having a flexible perspective is particularly important for biotherapeutics, it is important that choosing which technique to use is determined based on good science and begins with a physicochemical property assessment of the analytes, followed by subsequent alignment with the most appropriate retentive mode. An Earth-based figurative analogy is used to help highlight several popular LC techniques.

Favoritism in daily decision-making has a profound influence in our lives as humans. The world of analytical chromatography is no exception, especially in the regulated space with the associated pressures for quality and timeliness. We want something that works as best as it can and arrives at a functional situation as soon as possible.

In my field of quantitative bioanalysis, which includes biopharmaceuticals to a continually increasing extent (in contrast to the world dominated by small-molecule therapeutic candidates of around 30 years ago), there is a limited range of options for liquid chromatography (LC) operation, and these options may be broadly classified simply as polar and nonpolar. In quantitative bioanalysis, the well-trodden path is in applying non-polar, better known as reversed-phase (RP) operation. To use an analogy—if you think of chromatographic operation as the Earth, RP operation may be seen as lying roughly at the equator. The conditions are nice, the land is well-mapped, there are lots of other people with similar interests around, and everything seems to be well-established and characterized. If problems arise with RP operation, there is confidence about how chromatographers can troubleshoot these issues.

Then, moving north or south, we enter the polar regions. These regions are far less explored and relatively sparsely populated. A good dose of mystery persists about how to negotiate our way through these parts of the world and, moreover, how to thrive if we decide we wish to get comfortable there. The poles of the Earth analogy as would befit the analysis of polar analytes correspond to polar modes of chromatography, dominated by hydrophilic-interaction chromatography (HILIC) (1). By contrast, as mentioned, the regions closer to the equator correspond to the RP domain, where we can relax and focus on purely dispersive interactions while sipping piña coladas.

Moving between the poles and the equator, there are also fascinating areas corresponding to ion-exchange chromatography (IEC) and aqueous normal phase (ANP) chromatography, the bimodal function of silica-hydride–based column chemistries where the normal-phase modality can function simultaneously with the RP modality (2). The silica hydride surface can provide options in all chromatographic areas. Described in context with the Earth analogy, the snow-covered polar cap contrasts with the equatorial climes in terms of the diversity of available chemistries and the number of groups using that chromatographic mode. Figure 1 illustrates the analogy used in this article.

There is a great deal of comfort in RP operation. In chromatographic bioanalysis, users of LC–mass spectrometry (LC–MS) have roots in the small- molecule paradigm where they could so often use the RP modality with success. Hints of polar flavors could be added to stationary-phase chemistries, such as polar-embedded groups or polar endcapping, adjusting the selectivity and resolution as desired but without altering the fundamental mode of separation. To take the plunge into a different mode back in the days of small-molecule dominance, the options for unusual polar challenges were classical normal phase, which was sometimes used in tricky chiral applications, or HILIC. Between these, the only really practical option was HILIC, particularly given that it was the only option for which consumables and other support were readily available, and that it would not involve unfavorable operational aspects like system dedication because no dominant non-eluotropic solvent used in normal phase is water-miscible. Since approximately 2007, HILIC has modestly taken off in popularity in terms of the number of studies in the literature (3), and the increased usage of HILIC also corresponds to the increasing diversity of the stationary-phase chemistries available. As a result, the bioanalytical LC–MS community is becoming more familiar with the polar climes.

However, RPLC contains additional attributes that work in its favor in addition to the huge assets of familiarity, available phases, and knowledge base. RPLC is forgiving in various functional aspects, such as the tolerance—without chromatographic aberration—of a wide range of variability in the composition of injected solution. In analyzing bioanalytical extracts, decent sharp and symmetrical peaks, which are reflective of those from solution tests, may often be acquired in the crudest of extracts, and the columns maintain a good lifetime of more than one thousand injections. Additionally, the excursion in retention time with mobile-phase composition is significantly more linear and predictable than in normal-phase techniques.

All in all, reversed-phase LC is an old favorite and we can certainly understand the reasons why. Furthermore, the resistance to look toward other modalities strengthens in accordance with the affinity one feels towards reversed-phase. We have not only a wealth of different RP chemistries with advantageous polar incorporations available within the RP bracket, but we also have readily available ion-pairing reagents that can essentially make polar analytes nonpolar for the intended analytical endpoint of RPLC. However, ion-pairing reagents have several important disadvantages in LC–MS (4). The most notable of these disadvantages is its almost constant instrumental cleaning and concomitant downtime, short column lifetimes with permanent chemistry changes, and system dedication challenges. These considerations certainly impact the current biopharmaceutical analytical paradigm and will become more important in the future as LC–MS becomes more central as a bioanalytical platform, especially as instrument sensitivity continues to increase and as high-resolution MS technology becomes better aligned with the quantitative needs of chromatographers and is used more frequently.

Therefore, we can appreciate how RPLC is always attractive even though it is not always the ideal choice. Introducing ion pairing is a decision that, particularly in the world of LC–MS, should be carefully weighed against an alternative that may be suitable for the innate chemistry of the analyte molecular entities. In other words, a close look at physicochemical properties is certainly worthwhile.

A technique like HILIC may be appropriate more often than many may realize. In real projects involving new small-molecule drug candidates, we have frequently encountered that the presence of just one ionizable basic group is sufficient to enable HILIC retention, even if the remainder of the carbon skeleton bestows hydrophobicity. In antisense oligonucleotide work in the absence of ion pairing, our group found that HILIC worked emphatically well in quantitative bioanalytical LC–MS methods (5,6).

To put it simply, if an analyte or group of analytes is polar, there is a good chance that HILIC or another alternative to RPLC will work and provide the best results within the minimum time to complete. If the challenge involves hydrophobic analytes, then it is better to use RPLC as the first port of call in method development because that would be the best option. Because we know that “like dissolves like,” we can match polarity with chromato- graphic modality. With this logical basis, putting our proclivity for favorites aside, we can explore and become familiar with all areas of the chromatography world, even those that may seem frigid but are just waiting to be better characterized and have all their benefits revealed.

The author would like to thank Martin Squires for the illustration.

The opinions expressed are solely my own and do not express the views or opinions of my employer.

https://doi.org/10.1016/S0021-9673(00)96972-3

(4) M. Holcapek, K. Volnà, P. Jandera, L. Kolářová, K. Lemr, M. Exner, and A. Církva, 

(5) R. MacNeill, T. Hutchinson, V. Acharya, R. Stromeyer, and S. Ohorodnik, 

(6) P. Anand, M. Koleto, D. Kandula, L. Xiong, and R. MacNeill, 

 is with the Department of Bioanalysis at Labcorp Drug Development Inc. in East Millstone, New Jersey. Direct correspondence to: 

Using Earth as a figurative analogy, we explore the merits of flexibility when considering which liquid chromatography (LC) modality to use for any particular bioanalysis.

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