Read an Excerpt

Ambient Ionization Mass Spectrometry

The Royal Society of Chemistry

An Introduction to Ambient Ionization Mass Spectrometry

MARÍA EUGENIA MONGE AND FACUNDO M. FERNÁNDEZ

School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USA

1.1 Introduction

The introduction of desorption electrospray ionization mass spectrometry (DESI MS) by Cooks and coworkers in 2004 brought, for the first time, widespread attention to the concept of open-air surface analysis under ambient conditions. Contemporary with the disclosure of DESI, work carried in parallel by other research teams explored a similar philosophy in chemical analysis. Examples include the patent on the ion source named direct analysis in real time (DART) filed in December 2003, Shiea's work on open-air laser-based ion sources, and work by the Van Berkel group at Oak Ridge National Laboratory on surface sampling probes (SSPs) for direct sampling of thin-layer chromatography plates first published in 2002. DESI, DART, and other ambient MS techniques enabled an exciting new perspective on ways to perform both qualitative and quantitative chemical investigations on samples not typically amenable to direct MS analysis. As a bonus, direct analysis on native surfaces could be done, in most cases, without sample preparation.

Considering the pressures on modern analytical laboratories in terms of workload, turnaround time, and cost per sample, it is not surprising, in perspective, that ambient MS would rise so quickly to the forefront of analytical science. Our particular interest, as a research group, stemmed from our involvement in large surveys to study the quality of anti-infective medicines used to treat malaria. These surveys required that we rapidly screen large numbers (thousands) of drug tablets for the presence of falsified and other poor-quality medicines. Being able to simply hold a tablet in front of the atmospheric-pressure inlet of a high-resolution mass spectrometer while exposing it to an ionizing plasma and obtaining a pass/fail result in seconds almost seemed like magic. It goes without saying that initial experiments that our group did with DART before the technique was officially released, got us interested almost instantly. Typical chromatographic analysis for testing drug quality requires hours of sample preparation and at least tens of minutes for chromatographic analysis. The time savings with ambient MS instantly made this type of techniques a central step in our multitiered approach to detect and source falsified and other poor-quality pharmaceuticals. After more than 8 years using various ambient MS methods for falsified drug analysis, we can confidently say that this family of desorption/ionization techniques have definitely enabled unique analytical workflows that were not possible before 2004.

As skeptical scientists, we should still ask ourselves what is truly new with respect to ambient MS approaches. Will the excitement about ambient MS withstand the challenge of time? Will we see ambient MS approaches being routinely used in laboratories worldwide 20 years from now? The answers to these questions lie in the true usefulness of ambient MS. For analytical technologies to become widely adopted they have to offer capabilities that are sufficiently different from existing approaches. The key advantages of ambient MS approaches are in the format in which well-established ionization mechanisms are implemented to enable surface analysis. DART, for example, makes use of ionization mechanisms that predominantly follow atmospheric-pressure chemical ionization (APCI) pathways, but in an open-air format. This technique, however, has enabled experiments that are not easily performed by APCI. APCI, in its most common format, requires a liquid sample. This is not the case with DART, by which one can also readily examine solids and gases directly. Direct infusion APCI, which overcomes the chromatographic step, still requires sample dissolution and pre- and postanalysis rinsing of the tubing connecting the pump propeling the liquid into the ion source. The lack of any plumbing makes DART much more impervious to memory effects that arise from rapidly injecting samples with analytes in a widely varying concentration range. Therefore, the advantages that make DART MS attractive compared to direct infusion APCI MS are related to its minimum carryover, as all parts in contact with the sample are disposable, and its shorter analysis time, as there is no need for cleaning parts. More importantly, DART has also been shown to have advantages over APCI when compared side-by-side as ion sources in LC MS. For example, LC DART MS has shown less that 11% ionization suppression in the analysis of parabens in sewage-plant effluents in comparison to APCI, which showed ionization suppression that ranged between 20 and >90%. DART MS has enabled applications such as rapid forensic screening, rapid metabolomic profiling, rapid bacterial typing, chemical profiling of live animals to study pheromone-mediated behavior, fast screening of counterfeit drugs, low cost authentication of food products, and rapid detection of warfare agents, among others.

DESI, on the other hand, is in many ways complementary to DART (Table 1.1) and makes use of desorption mechanisms that involve a continuous solid–liquid extraction process, while capitalizing on the known ionization mechanisms of ESI. As with DART, DESI has enabled applications that are not possible by ESI. Examples include imaging of tissues in reactive mode, in vivo imaging of secondary metabolites, intraoperative lipid profiling of brain tumor tissue sections, direct detection of chemical warfare agents, imaging of counterfeit pharmaceuticals from developing-world countries, clustering based on sample composition, and imaging products of heterogeneous model prebiotic reactions on the surface of minerals, to name a few examples. We have chosen the following two case studies to showcase in more detail unique advantages brought by DART and DESI, the two flagships of ambient MS approaches:

(i) The work by Musah et al. illustrates the effectiveness of DART MS in forensic drug chemistry, demonstrated by the detection of synthetic cannabinoids in herbal blends. Detection and identification of these compounds is challenging given the wide range of active ingredients, and the variety of botanical matrices in which they are found. In addition, these substances are not part of routine drug screens, and metabolites in urine would not show positive for marijuana use. To make this application even more challenging, none of the synthetic cannabinoids trigger a positive drug test using standard immuno-logical screening procedures, and they are particularly problematic for screening methods that rely on a library search for identification because these substances are rarely included in standard databases. Additionally, the matrix in which synthetic cannabinoids are found can be comprised of several types of plants, making their detection even more arduous. This work shows that DART MS is capable of overcoming these difficulties and identifying this type of compound with a high degree of confidence without using a database. Plant leaves doped with the AM-251 and JWH-015 synthetic cannabinoids can be analyzed by simply holding the leaves with tweezers between the ionization source and the mass-spectrometer inlet. Despite the complex mass-spectral profiles given by the plant matrices, DART MS was capable of identifying the target compounds without the need for extraction or other sample preparation steps. Ionization suppression did not significantly affect analysis since 300 µg of cannabinoid were easily detected within an excess of background matrix. Unique advantages of DART MS illustrated in this case study, and that can be extended to other applications include: (i) no need for solvents, extractions, sample processing, or preparation steps; (ii) resulting spectrum produced in seconds; (iii) high-throughput analysis with no carryover between samples, and (iv) no plant matrices interference in the detection of target compounds. In contrast, comparative analysis performed by gas chromatography/mass spectrometry (GC/MS) required approximately 3 days to be completed. Analyte solvent extraction is usually undesirable given the drawbacks associated with additional sample preparation steps such as higher blanks, low recovery, and decreased sample throughput. Given that illicit drug manufacturers have demonstrated the capability to rapidly modify the components and formulations that they market, instrumentation and methodologies that can readily identify the presence of illicit substances are highly desirable.

(ii) The study by Wu et al. is another example highlighting how ambient techniques can tackle challenging applications. This work illustrates the capability of reactive DESI to detect cholesterol in human serum and in rat brain tissues with high sensitivity and selectivity by incorporating betaine aldehyde into the spray solvent. The experiment combines a chemical derivatization in situ that takes place in the short timescale of the solvent extraction–ionization–detection process to efficiently detect a nonpolar compound. A rapid and selective nucleophilic addition occurs at the spot being sampled, generating a positively charged hemiacetal, which allows the detection of a low proton affinity analyte that is hardly ionized by common soft ionization techniques. The capability of quantitative analysis of free cholesterol in human serum was demonstrated using the standard addition method. Serum calibration solutions, spiked with isotopically labeled cholesterol-d7 as internal standard, were manually spotted on a glass slide and analyzed with reactive DESI MS. Matrix interferences were mitigated due to the high selectivity of the nucleophilic addition reaction. The performance of this quantitation method was comparable to GC/MS and ESI MS, but accomplished in a shorter timescale. A detection limit of 1 ng was achieved with reactive DESI when a solution of 1 µg mL-1 was spotted onto the surface. In addition, using 65 ppm betaine aldehyde in acetonitrile : water : dimethylformamide (8 : 3 : 1), cholesterol in rat brain tissue was imaged under ambient conditions giving a full 2D image at a 200 µm pixel size resolution within an hour. This ambient MS technique provides unique advantages for cholesterol detection in comparison to colorimetric extraction assays, and traditional hyphenated techniques in terms of high-throughput analysis, as there is no need for sample pretreatment such as derivatization, extraction or other timeconsuming steps. If the sample amount is not a critical limitation, this approach can enable successful quantitation of cholesterol in biological fluids. Similarly, when high lateral resolution is not needed, reactive DESI provides imaging capabilities for mapping low-polar compounds in biological tissues under atmospheric pressure and using a soft ionization technique with no need for matrix addition.

With so many new ionization techniques being reported since 2004, distinguishing ambient ionization techniques from more conventional atmospheric-pressure ionization techniques can help delineate the different applications that are best paired with each approach. To this purpose, we propose a set of basic characteristics that should be present in techniques to be part of the "ambient ionization/sampling" MS field. First, ambient MS techniques should be able to carry ionization in the open air. This is a critical feature when examining objects of unusual shape or size such as plants, solid phase extraction fibers/bars, tablets, fabrics, etc. in direct analysis applications. Direct surface analysis capability is another key attribute of ambient MS techniques. This is particularly useful for surface analysis of solids, avoiding many, if not all sample preparation steps typically required in MS-based chemical analysis. Ambient MS ion sources are easily swappable in most types of mass spectrometers fitted with atmospheric-pressure interfaces. No modification to the ion transfer optics or the vacuum interface are generally needed for ambient MS operation, with the exception, in some cases, of suction interfaces to reduce the gas load and prevent damage to the vacuum system. It goes without saying that ambient MS techniques should generate ions without significant fragmentations, as is the case with their atmospheric-pressure counterparts.

As in ambient MS, many newly reported ionization approaches also strive to incorporate sample-preparation steps into the ionization process or analyze samples in its native form. Examples include paperspray ionization, extractive electrospray ionization (EESI), and fused droplet ESI (FD-ESI). Sometimes these approaches are bundled into the ambient MS field, but not being surface-analysis techniques, we argue that this may not be correct. Paperspray ionization incorporates simple chromatographic separation and/or solid-phase extraction processes so they occur simultaneously with the ionization process, allowing direct analysis of dried biofluid samples. EESI and FD-ESI incorporate a continuous liquid–liquid extraction step into the ionization process, leading to increased salt tolerance than when compared with ESI. This feature is useful for the simplified extraction of trace analytes, such as melamine, from complex samples such as milk. Extensions of the paperspray concept can be found in the recently described "tissue-spray", and leaf-spray techniques. In these cases it is possible to perform electrospray ionization of tissue components directly by wetting the sample with solvent. The sample is usually cut to have a sharp end from which to initiate the electrospray process. Paperspray, EESI/FD-ESI, tissue-spray, leaf-spray and similar techniques are best classified as direct ionization techniques more closely related to ESI than to DESI and DART.

A number of review articles and tutorials are available on the topic of ambient ionization and ambient imaging. The classification of the various ambient MS techniques in subclasses varies in these reviews, with a certain degree of overlap. Our group and others have classified ambient MS techniques based on their intrinsic desorption/ionization mechanisms, but these divisions are sometimes debatable. This is the case when several concurrent desorption/ionization mechanisms occur. The subdivisions that we propose are as follows:

• One-step techniques where desorption occurs by solid–liquid extraction followed by ESI, APPI, sonic spray, or CI ion production mechanisms.

• One-step plasma-based techniques involving thermal or chemical sputtering neutral desorption followed by gas-phase chemical ionization.

• Two-step techniques involving thermal desorption or mechanical ablation in the first step followed by a second, separate step where secondary ionization occurs.

• Two-step techniques involving laser desorption/ablation followed by an independent secondary ionization step.

• Two-step methods involving acoustic desorption approaches.

• Multimode techniques combining two or more ambient MS techniques.

• One-of-a-kind techniques that make use of other principles for desorption or ionization that do not belong to any of the previous categories.

Table 1.2 describes the techniques that fall into the aforementioned division. It is clear from this table that not all reported techniques are truly different from each other, sometimes the differences simply being a specific set of experimental conditions (such as type of laser used, flow regime, etc.). Distinguishing between true innovation and the simple rebranding of already-reported techniques continues to be a challenge. For these reasons, authors are strongly discouraged to give existing techniques new names. In order to provide a general overview of the most common ambient MS approaches, a brief description of them is given here.

1.1.1 One-step Techniques where Desorption occurs by Solid–Liquid Extraction followed by ESI, APPI, Sonic Spray, or CI Ion Production Mechanisms

Two groups of techniques, and desorption atmospheric-pressure photo ionization (DAPPI) fall in this category. The first group includes DESI and its variants such as reactive DESI, transmission mode-DESI (TM-DESI), de sorption ionization by charge exchange (DICE). It can be tempting to include easy ambient sonic-spray ionization (EASI) in this group, but it must be taken into account that the ionization mechanisms in DESI and EASI are different. The second group is based on the formation of liquid micro-junctions (LMJs), and comprises LMJ-surface sampling probe (LMJ-SSP), liquid-extraction surface analysis (LESA), and nanospray desorption electrospray ionization (nano-DESI).

---

Excerpted from Ambient Ionization Mass Spectrometry by Marek Domin, Robert Cody. Copyright © 2015 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
All rights reserved. No part of this excerpt may be reproduced or reprinted without permission in writing from the publisher.
Excerpts are provided by Dial-A-Book Inc. solely for the personal use of visitors to this web site.

---