Review pubs.acs.org/CR
Mass Spectrometry: Recent Advances in Direct Open Air Surface Sampling/Ionization María Eugenia Monge,†,# Glenn A. Harris,‡,# Prabha Dwivedi,†,# and Facundo M. Fernández*,† †
School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332, United States Department of Biochemistry and the Mass Spectrometry Research Center, Vanderbilt University, Nashville, Tennessee 37235, United States
‡
2.3.3. Atmospheric Pressure Thermal Desorption-Secondary Ionization (AP-TD/SI) 2.3.4. Probe Electrospray Ionization (PESI) 2.4. Two-Step Laser-Based Desorption Ablation Techniques 2.4.1. Laser-Based Hybrid Techniques Coupled to ESI or Plasma Ionization 2.4.2. Laser Electrospray Mass Spectrometry (LEMS) 2.4.3. Laser Ablation Atmospheric Pressure Photoionization (LAAPPI) 2.4.4. Laser Ablation Sample Transfer 2.5. Acoustic Desorption Techniques 2.5.1. Laser-Induced Acoustic Desorption (LIAD) 2.5.2. Radiofrequency Acoustic Desorption Ionization (RADIO) 2.5.3. Surface Acoustic Wave-Based Techniques 2.6. Multimode Techniques 2.6.1. Desorption Electrospray/Metastable-Induced Ionization (DEMI) 2.7. Other Techniques 2.7.1. Rapid Evaporative Ionization Mass Spectrometry (REIMS) 2.7.2. Laser Desorption Ionization (LDI) 2.7.3. Switched Ferroelectric Plasma Ionizer (SwiFerr) 2.7.4. Laserspray Ionization (LSI) 3. Remote Sampling 3.1. Nonproximate Ambient MS 3.2. Fundamentals of Neutral/Ion Transport 3.3. Transport of Neutrals 3.4. Transport of Ions 4. Future Directions Author Information Corresponding Author Author Contributions Notes Biographies Acknowledgments References
CONTENTS 1. Scope of this Review 2. Ambient Ionization Techniques 2.1. Solid−Liquid Extraction-Based Techniques 2.1.1. Desorption Electrospray Ionization (DESI) 2.1.2. Desorption Ionization by Charge Exchange (DICE) 2.1.3. Easy Ambient Sonic-Spray Ionization (EASI) 2.1.4. Liquid Micro Junction Surface Sampling Probe (LMJ-SSP) 2.1.5. Liquid Extraction Surface Analysis (LESA) 2.1.6. Nanospray Desorption Electrospray Ionization (nanoDESI) 2.1.7. Desorption Atmospheric Pressure Photoionization (DAPPI) 2.2. Plasma-Based Techniques 2.2.1. Direct Analysis in Real Time (DART) 2.2.2. Flowing Atmospheric-Pressure Afterglow (FAPA) 2.2.3. Low Temperature Plasma (LTP) & Dielectric Barrier Discharge Ionization (DBDI) 2.2.4. Chemical Sputtering/Ionization Techniques 2.3. Two-Step Thermal/Mechanical Desorption/ Ablation (Non-Laser) Techniques 2.3.1. Neutral Desorption Extractive Electrospray Ionization (ND-EESI) 2.3.2. Beta Electron-Assisted Direct Chemical Ionization (BADCI)
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1. SCOPE OF THIS REVIEW This review discusses advances in the field of ambient sampling/ionization mass spectrometry (“ambient MS” for short) with a focus on mechanistic and instrumentation studies. It also provides a discussion of the numerous ambient MS applications reported in the period ranging from January 2011 to June 2012. Literature searches through PubMed and SciFinder Scholar electronic databases were performed independently by all authors. The keywords used in the searches included the technique names (Table 1) with and without their corresponding abbreviations, followed by the words “ambient mass spectrometry” or “mass spectrometry”. Because of the large number of published papers in this area, we have not attempted to make this review article all-inclusive, but rather to select a number of key reports that illustrate capabilities of this family of ionization techniques for MS, and the main trends in the field through some of the most demanding applications. We chose to focus on fundamental and mechanistic aspects in conjunction with innovative instrumentation developments rather than on an exhaustive coverage of all applications described in the current literature. A number of excellent review articles and tutorials are already available on the topic of ambient ionization and imaging,1 and the reader is kindly directed to those to supplement the material presented here. A key goal of this review is to critically delineate future directions and to identify ongoing challenges. Most importantly, we have attempted to provide a balanced coverage of the field, avoiding over-referencing of our own work. The first widespread attention to the concept of ambient ionization/sampling prior to MS analysis was the introduction of desorption electrospray ionization (DESI) by Cooks and coworkers in 2004.2 This article presented an exciting new perspective on performing both qualitative and quantitative chemical analysis by MS. The central idea was that the surface of real-life objects such as a piece of leather, a nitrile glove, a plant seed cut in half, an intact tomato, the tip of a finger, etc. could be directly examined by MS without extraction of the molecules of interest or pretreatment of the object itself. The photograph showing Dr. Takáts performing DESI analysis of an intact flower (Figure 1) as presented in the original Science article, has become iconic of the philosophy embedding a typical ambient MS experiment: “surface analysis in the open air without sample preparation”. Contemporary with the development of DESI was the work of several other researchers exploring a similar chemical analysis philosophy. Examples include patents on the ion source named direct analysis in real time (DART) filed in December 2003,3 Shiea’s work on laser based ion sources,4 and work by the Van Berkel group at Oak Ridge National Laboratory on surface sampling probes (SSPs) for direct thin layer chromatography sampling5 first published in 2002. The excitement about the new analysis opportunities offered by the field of ambient sampling/ionization has triggered a renaissance in the development of new ion generation approaches, and their application to real-life analytical and diagnostic challenges. As a downside, this has effectively turned the keyword “ambient” into a buzzword many times improperly used. To distinguish ambient ionization approaches from atmospheric pressure ionization techniques, we propose a set of basic characteristics that should be present in techniques to be included in the “ambient ionization/sampling” MS field
Figure 1. DESI analysis of a geranium flower using a methanol/water spray mixture. This experiment allowed interrogating the surface of this fragile sample in a spatially resolved fashion. In the background we see a linear ion trap mass spectrometer whose inlet was modified to allow remote sampling. (Reprinted with permission from ref 2. Copyright 2004 American Association for the Advancement of Science.)
(Chart 1). Ambient MS techniques should enable (a) ionization in the absence of enclosures such as those typically Chart 1 Ambient sampling/ionization implies the following.
found in electrospray ionization (ESI), atmospheric pressure photoionization (APPI), atmospheric pressure chemical ionization (APCI), or atmospheric pressure matrix-assisted laser desorption ionization (AP-MALDI) sources. This feature is critical when examining objects of unusual shape or size that could not be easily fitted inside of an ion source enclosure or that would be critically damaged when placed under a vacuum. In other words, the technique should operate in the ambient environment or open air. This characteristic not withstanding, commercial versions of ambient MS ion sources many times employ enclosures for safety reasons (i.e., prevent exposure to high power laser radiation, heating elements or high voltage) but allow sufficient flexibility so that various objects can be still sampled directly. (b) Ambient MS techniques can perform direct ionization avoiding sample preparation steps typically used in MS-based chemical analysis. The steps avoided include solid-phase extraction, preconcentration, liquid−liquid extraction, off-line derivatization, dissolution, chromatographic or electrophoretic separations. An interesting trend in the field of ambient MS is to build one or many of these steps into the sampling/ionization process itself, avoiding extra laboratory 2270
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Table 1. List of Ambient MS Techniques, Their Abbreviations, and Relevant References to First Reportsa abbreviation
first report
figure
Solid−Liquid Extraction-Based 1, 2 DICE desorption ionization by charge exchange 16 LMJ-SSP liquid micro junction-surface sampling probe 4 LESA liquid extraction surface analysis
DESI EASI DAPPI
desorption electrospray ionization easy ambient sonic spray ionization desorption atmospheric pressure photo-ionization
2 100 138
DART FAPA ASAP
direct analysis in real-time flowing atmospheric pressure afterglow atmospheric solids analysis probe
161c 225b 150
5 8
LTP
low-temperature plasma probe
233
9
DAPCI
desorption atmospheric pressure chemical ionization dielectric barrier discharge ionization
168
DBDI
ND-EESI BADCI
ELDI MALDESI LAESI LADESI LDESI RADIO LIAD/ESI
DEMI
REIMS LDI a
name
abbreviation
Plasma-Based DCBI PADI APTDI HAPGDI
name
desorption corona beam ionization plasma assisted desorption ionization atmospheric pressure thermal desorption/ ionization helium atmospheric pressure glow discharge ionization plasma pencil atmospheric mass spectrometry LTP
PPAMS LTP 232 ambient ambient microhollow cathode discharge ionization MHCD Two-Step Thermal/Mechanical Desorption/Ablation (Non-Laser) neutral desorption extractive 363 AP-TD/SI atmospheric pressure thermal desorption-secondary electrospray ionization ionization beta electron-assisted direct chemical 259 PESI probe electrospray ionization ionization Two-Step Laser-Based Desorption Ablation electrospray-assisted laser desorption 4 LEMS laser electrospray mass spectrometry ionization matrix-assisted laser desorption 284 LD-APCI laser desorption atmospheric pressure chemical electrospray ionization ionization laser ablation electrospray ionization 275 10 IR-LAMICI infrared laser ablation metastable-induced chemical mass spectrometry ionization laser-assisted desorption electrospray 279 PAMLDI plasma assisted multiwavelength laser desorption ionization ionization laser desorption electrospray ionization 280 LAAPPI laser ablation atmospheric pressure photoionization Acoustic Desorption radio-frequency acoustic desorption 324 LIAD/ laser-induced acoustic desorption/atmospheric and ionization APCI pressure chemical ionization laser-induced acoustic desorption321a 13 SAWN surface acoustic wave nebulization electrospray ionization Multimode desorption electrospray/metastable332 induced ionization Other Techniques rapid evaporative ionization mass 333 SwiFerr switched ferroelectric plasma ionizer spectrometry laser desorption ionization 334 15 LSI laserspray ionization
first report 96 5 125a
figure
3
250 146 148 149 234 160
260 263
282 278 276
11
186 314
12
323 325
14
335 364
Techniques in italics are available commercially.
desorption mechanisms that follow a continuous solid−liquid extraction process, while capitalizing on the known ionization mechanisms that follow ESI. However, DESI MS has enabled applications that are not possible by ESI or any off-line solid− liquid extraction procedure combined. Examples include imaging of tissues without vacuum or exogenous matrix deposition,7 in vivo imaging of secondary metabolites on algae tissues,8 nonproximate detection of explosives,9 direct detection of chemical warfare agents,10 investigation of large libraries of counterfeit pharmaceuticals from developing world countries,11 and clustering based on sample composition,12 to name a few high-impact examples. Ambient MS techniques are bound to establish themselves as key tools for screening, pass/ fail analysis, fingerprinting, and native sample imaging applications. As achieving the analysis of native samples is within the goals of ambient MS, a section describing remote sampling techniques has been included in the present review. The capability to transport distantly generated ions and
steps. (c) Ambient MS techniques are generally interfaceable to most types of mass spectrometers fitted with atmospheric pressure interfaces. In the same way that AP-MALDI made MALDI analysis available to ESI users,6 ambient MS users can take advantage of techniques such as DART and DESI on the same instrument by using swappable ion sources. In these scenarios, no modification to the ion transfer optics or the vacuum interface is needed. (d) Ambient MS techniques focus on generating ions softly, with amounts of internal energy deposited equal or lower than those in their atmospheric pressure counterparts. The skeptical reader may ask what is truly new with respect to ambient MS approaches. This is a fair question considering that the field is now past its initial hype period and should be finding its own analytical niche. We argue that the key advantages of ambient MS approaches are in the format in which known ionization mechanisms are implemented to enable surface analysis. For example, DESI makes use of 2271
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ions at atmospheric pressure when an analyte solution is pumped through a small capillary in a way that the liquid is dispersed as a fine mist of droplets into an electric field.20 The spraying process is often assisted by a nebulizing gas. The electric field gradient created between the tip of the spraying capillary and the inlet of the mass spectrometer by application of high voltage (2−5 kV) produces charge separation at the surface of the liquid.21 The solution projects from the capillary tip as a “Taylor cone” reaching the point at which Coulombic repulsion of the surface charge is equal to the surface tension of the solution (Rayleigh limit), and droplets that have an excess of positive or negative charge detach from its tip. In positive ion mode, the main contributors to the net droplet charge are protons generated at the sprayer tip interface through water oxidation.22 The size of the micrometer droplets is reduced by solvent evaporation and fission events to highly charged nanodroplets, which finally lead to the formation of gas-phase charged analyte ions.20b,23 The two major mechanisms that have been suggested to explain the ion release are the charge residue model (CRM)24 and the ion evaporation model (IEM).25 The CRM suggests that the analyte in Rayleighcharged nanodroplets retains some of its droplet’s charge as the last solvent shell evaporates. The IEM assumes that small solvated ions are ejected from droplet surfaces when Coulombic repulsion overcomes the liquid’s surface tension and solvation forces. The CRM is usually accepted for large globular molecules, whereas the IEM applies for low molecular weight analytes. Recently, a chain ejection model has also been proposed for disordered polymers that are partially hydrophobic and capable of binding excess charge carriers, such as unfolded proteins.26 APCI was first described using a 63Ni radiation source, which was then replaced by a corona discharge.27 In contrast to ESI, ionization occurs in the gas phase, and in the ionization area there is high voltage (∼2−5 kV) applied to a needle point to promote and maintain a corona discharge. Primary electrons produced in the discharge ionize the N2 in air, and ambient gases, forming radical cations in the positive ion mode.27a These initiate a sequence of ion/molecule reactions that lead to the formation of hydronium ion−water clusters H3O+(H2O)n, which become the main reagent to ionize the vaporized neutral analytes through proton transfer reactions.28 In negative mode, oxygen is needed to start the ionization cascade and leads to proton abstraction reactions from hydroxylated water clusters. The reactions are driven by the gas-phase acidity and basicity of the analytes and the reagent ions. The reagent ion cloud that promotes the ionization of the analyte is favored at atmospheric pressure due to the higher number of collisions. Additional ionization reactions involve charge transfer, and cation attachment in positive ion mode, and electron capture and anion attachment in negative ion mode. Different ionization reagents as well as dopants introduced in the discharge area can be used to facilitate ionization of analytes with different polarities through these alternative channels. If the analyte is initially dissolved in a solution, then this is nebulized into a heated region at atmospheric pressure where both analyte and solvent are vaporized using nitrogen or air as nebulizing gas.
neutrals to the mass spectrometer for their analysis has further broadened the applications of ambient MS.
2. AMBIENT IONIZATION TECHNIQUES The recent upsurge in interest for new ionization approaches has blurred the boundaries of what is traditionally considered to be a surface sampling/ionization ambient MS technique. Many new ionization approaches, although not focused on surface analysis, also strive to incorporate sample preparation steps into the ionization process or analyze samples in its native form. Examples of ionization techniques with built-in sample preparation steps include paperspray ionization,13 extractive electrospray ionization (EESI),14 and fused droplet ESI (FDESI).15 Paperspray ionization incorporates adsorption chromatography and/or solid phase extraction steps directly into the ionization process, allowing direct analysis of dried biofluid spots for therapeutic drug monitoring with whole blood samples, and detection of illicit drugs in raw urine, for example.13b EESI and FD-ESI incorporate a continuous liquid− liquid extraction step into the ionization process, leading to a much higher salt tolerance than with ESI, which has enabled direct analysis of trace analytes, such as melamine, in complex samples such as milk.14b This type of analysis would normally require extensive sample pretreatment. More recently, direct analysis of aerosol drugs by nano-EESI has been demonstrated making use of a continuous solid−liquid extraction procedure in the ionization plume.16 Examples of other ionization techniques that do not necessarily fall under the umbrella of ambient MS, but that focus on the analysis of samples in their native form, are the recently described “tissue-spray”17 and leafspray techniques.18 Both tissue and leaf spray enable electrospray ionization of tissue molecular components by wetting the sample with a solvent, which acts as the extractant, followed by direct ionization of the extract from the plant leaf or tissue, cut to a sharp point. Paperspray, EESI/FD-ESI, tissue-spray, leafspray and similar techniques (such as toothpick-ESI19) are examples of direct ionization approaches for MS. However, we do not include them in this review as part of the ambient sampling/ionization technique family because their focus is not on surface analysis, thus departing from the trend set in the initial Science article. Our group1c,n and others1l,m have traditionally classified surface sampling ambient MS techniques based on the intrinsic desorption/ionization mechanisms grouped in Table 1. Following the present status of the field, we propose the following seven categories: (1) One-step techniques where desorption occurs by solid−liquid extraction processes dynamically followed by ESI, APPI, sonic spray, or CI ion production mechanisms, (2) One-step plasma-based techniques involving thermal or chemical sputtering desorption of neutrals working in parallel with chemical ionization mechanisms, (3) two-step techniques involving thermal desorption or mechanical ablation followed by secondary ionization, (4) two-step techniques involving laser desorption/ablation followed by secondary ionization, (5) two-step acoustic desorption methods, (6) multimode techniques involving two of the principles in previous categories, (7) one-of-a-kind techniques that do not fit into previous categories and that are part of or offer potential for becoming useful part of the ambient MS toolbox. As many ambient ionization techniques follow ESI or APCI ion production mechanisms, their fundamentals are briefly described. ESI converts solution-phase analytes into gas-phase
2.1. Solid−Liquid Extraction-Based Techniques
2.1.1. Desorption Electrospray Ionization (DESI). DESI Fundamentals. In the most simplistic explanation, DESI is a modified version of pneumatically assisted ESI29 where a sprayer probe is attached to an adjustable XYZ positional 2272
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Figure 2. Home-built DESI ion source (reprinted with permission from ref 361. Copyright 2007 American Chemical Society).
between 5−30°. The DESI sprayer is usually held fixed in position with the samples being placed manually by hand or automatically via a moving stage or rail system. The computercontrolled continuously moving stage approach is required for mass spectrometry imaging (MSI) experiments. A raster pattern or unidirectional scans are used with the latter approach yielding higher quality images.31 As in ESI, a high voltage connection is required to produce the primary electrically charged droplets that are emitted from the spray nozzle. The high voltage connection is either made directly to the solvent via an upstream zero-dead volume metal tee connection or an external alligator clip attached to the sprayer assembly if electrically conductive. Solvent flow rate varies depending on its composition, analyte, and surface properties, but typically falls between 1.5 and 10 μL min−1. The surface processes occurring at the area impacted by the solvent jet have been thoroughly investigated for DESI in order to explain how liquid extraction, desorption, and ionization occur. Computational fluid dynamic (CFD)32 and surface charge density imaging33 studies strongly support that the DESI process proceeds via a “droplet pick-up” mechanism that can be explained by conceptualizing it as if consisting of three consecutive stages. The first stage begins when the incident spray solvent contacts the sample surface forming a thin liquid film. The film is not confined to just the impact region as it will elongate outward in the direction of the spray (the spray footprint).34 The spray footprint includes the spray impingement region,35 which is not only the impact zone, but the solvent front and secondary droplets that extend beyond the solvent front. MSI applications where high spatial resolution is desirable must minimize the size of the impingement region by changing the previously mentioned variables to reduce the effective spray spot size. In the second DESI step, the liquid solvent film formed on the surface assists in the extraction of the analyte. Changing the organic/aqueous solvent ratio is the most straightforward approach to improve analyte solubility with the most common solvent system consisting of 50−70% organic solvent (methanol or acetonitrile) with 90%. Visualization of the spatial dimensions of the surface area probed by DART during at-an-angle experiments has shown sampling spots ∼5 mm in diameter.185 For applications requiring improved lateral spatial resolution, such as MSI or HPTLC scanning, coupling of DART to laser ablation techniques has been the instrumental configuration with the highest performance, at the cost of increased instrument complexity. The first report used a home-built DART ion source coupled to IR laser ablation in an approach named IR laser ablation metastable induced chemical ionization (IRLAMICI, see section 2.4.1). In a similar configuration, a plasma assisted multiwavelength (1064, 532, and 355 nm) laser desorption ionization mass spectrometry (PAMLDI MS) system was applied to the analysis of low molecular weight compounds through combination with TLC. 186 These approaches promise to complement laser ablation-ESI approaches (section 2.4.1) nicely, offering a wider range of ionizable analyte polarities and ambient MSI capabilities. In a recent report, laser ionization time-of-flight mass spectrometry (LI-TOFMS) was coupled to a DART ion source to coin a new technique named laser ionization metastable ionization time-of-flight mass spectrometry (LI-MI-TOFMS), as an alternative to laser induced breakdown spectroscopy (LIBS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP MS) for solids elemental analysis.187 A series of metallic samples from the National Institute of Standards and Technology (NIST 494, 495, 498, 499, and 500) and a pure carbon target were investigated by LI-MI-TOFMS in open air, which was found to be superior to LIBS in some cases. Laser pulse energies between 10 and 200 mJ at the second harmonic (532 nm) of an Nd:YAG laser were applied in the experiment. Applications. A growing number of DART applications has been reported in the 2011−2012 period when compared to previously reviewed ambient MS application work.1c This growth reflects an ongoing trend where plasma-based ambient 2285
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Figure 8. (a) Diagram of the original, pin-to-plate FAPA configuration with the discharge chamber constructed from Teflon. A hole in the plate anode allows discharge species to flow into the afterglow sample-introduction region. (b) Depiction of the new pin-to-capillary geometry FAPA with a Mykroy ceramic discharge cell. Discharge species flow into the sampling region through the anode capillary. Reprinted with permission from ref 231. Copyright 2011 American Chemical Society.
DART is also being extensively applied in the field of forensics for the direct examination of archival printing and writing papers,211 methamphetamine from syntheses or habitual smoking deposited on household surfaces during decontamination procedures,212 bank dye security pack and pepper spray chemicals on clothing,213 organic dyes in rare cultural heritage materials,214 sexual assault non-DNA trace evidence,215 synthetic cannabinoids in herbal samples,216 and nitro-organic and peroxide explosives in latent fingermarks.217 Since the first reports in 2010,218 DART applications in the field of metabolomics have shown a steady growth. Examples in the 2011−2012 period include beer authentication by applying partial least-squares discriminant analysis (PLS-DA), linear discriminant analysis (LDA), and artificial neural networks with multilayer perceptrons (ANN-MLP) to metabolomic profiles,219 breast cancer detection by combining DART and NMR metabolomic data,220 and high throughput screening of Arabidopsis thaliana mutant seeds.221 Other studies have focused on the coupling of DART to high resolution MS for identifying specific metabolites or families of metabolites from plants,170e,222 marine invertebrates,223 fruit juice,224 and propolis.170b 2.2.2. Flowing Atmospheric-Pressure Afterglow (FAPA). In the peer-reviewed literature, FAPA (Figure 8) was first described by Andrade et al. as the flowing afterglowatmospheric pressure glow discharge (FA-APGD) ion source225 and was first introduced at Pittcon 2005, months after DART. To the untrained eye, DART and FAPA may seem identical, but in their typical configurations these two techniques are different in many key instrumental aspects: (a) plasma species in FAPA are not filtered by any electrodes before interaction with the sample, leading to a more diverse array of plasma species, (b) FAPA is operated in the current-controlled glowto-arc i−V regime (∼25 mA) whereas DART is typically operated at lower currents, and (3) heating of the gas stream in FAPA is achieved through Joule heating within the electrical discharge and not by an external heater. These instrumental differences lead to more subtle, but equally important, differences.226 For example, a “DART-like” ion source was observed to produce protonated water clusters (H2O)nH+ with n = 2−7 as the reagent ions. FAPA, on the other hand, showed N2+, NO+, O2+ and (H2O)nH+ with n = 2−3 as the main species. The presence of N2+, NO+, O2+ in the FAPA background indicates that charge transfer reactions could easily occur with this technique, offering a wider range of ionization capabilities, but that could also somewhat complicate the mass spectrum.
In a recent study by Hietje et al.,227 the APGD and afterglow of a helium FAPA ionization source were examined by spatially resolved optical emission spectroscopy under a variety of operating conditions, including discharge current, electrode polarity, and plasma-gas flow rate. A special FAPA cell made of quartz was employed for these purposes. These studies indicated that, in the point-to-plane geometry, an appreciable amount of atmospheric water vapor, N2, and O2 can flow through the hole in the plate electrode into the discharge chamber, producing a significant amount of reagent ions that can lead to analyte oxidation and reduced sensitivity. Maximum rotational temperatures (∼1100 K) were found at the pin cathode. In terms of instrumentation development, FAPA has been used as an ion source for GC MS228 and miniaturized to some extent as the “miniFAPA” ion source.229 The mini-FAPA was tested as an interface for coupling capillary electrophoresis (CE) to MS by spraying the CE effluent using a sonic spraytype nebulizer.230 The mist from the sprayer was entrained with the FAPA plasma at a ∼45° angle, enabling independent control of ionization and separation voltages. In a recent report, a change in FAPA configuration from a point-to-plane to a point-to-capillary geometry and the use of machinable ceramics to build the discharge chamber was proposed (Figure 8).231 This new FAPA geometry was shown to greatly improve performance, with background signals in positive- and negativeionization modes reduced by 89% and 99%, respectively, due to the absence of fluorinated polymers in the building materials. In this geometry, analyte oxidation was also greatly reduced following a reduction in the amount of atomic oxygen generated from molecular diffusion of molecular oxygen into the discharge chamber. 2.2.3. Low Temperature Plasma (LTP) & Dielectric Barrier Discharge Ionization (DBDI). The initial description of dielectric barrier discharges (DBDs) for the ionization of organic compounds received the name DBDI232 (Table 1). This embodiment described a point-to-plane configuration where the sample substrate was used as the dielectric. An annular DBD configuration was later reported which received the name LTP.233 The annular configuration has the advantage that the operator can direct the plasma toward specific parts of the surface sample. A more recent configuration, described as the “plasma pencil atmospheric mass spectrometry” (PPAMS) LTP,234 used a design very similar to the original LTP experiment. PPAMS LTP was applied to the detection of key micronutrients such as zinc, iron, folate, vitamin A, and iodine in porcine plasma. LTP ionization has recently been combined 2286
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Figure 9. Schematic showing configuration of the LTP probe for ambient ionization MS. Reprinted with permission from ref 240. Copyright 2011 American Chemical Society.
by PCA allowing distinction of gram-positive and gramnegative bacteria and 11 out of 13 Salmonella strains.242 Related work has shown that unsaturated fatty acids and esters can be oxidized by in situ-generated ozone during ionization using LTP.243 Fragmentation patterns of the resulting oxidation products can be used to assign the double bond positions. Spatially resolved measurements with modest lateral resolution are also possible with LTP. Examples include depth profiling of thin layer coatings of electronics244 and imaging of Chinese paintings and calligraphy.245 Great progress has been recently made in the elucidation of LTP ionization mechanisms by means of plasma spectroscopic diagnostics.165,246 Imaging of helium metastable densities in the afterglow of a DBD using collisionally assisted laser-induced fluorescence by Farnsworth et al. indicated that metastable atom densities increased substantially when a glass slide was placed 10.0 mm from the discharge capillary in a geometry typical for desorption-ionization experiments, and that addition of hydrogen to the discharge severely quenched the metastable state leaving it virtually undetectable.165 Although metastable helium atoms play a central role, either as direct reagents involved in analyte Penning ionization processes, or as the indirect precursors for other highly excited helium species intervening in the ionization pathways, the observed quenching following hydrogen addition did not correlate with a decrease in sensitivity, but exactly the opposite. This finding suggests that additional reactive species are formed upon H2 addition to the plasma. Hieftje, Cooks et al. have recently investigated the role of reactive ionic species in DBD plasmas through spectroscopic measurements.246 The helium dimer He2+ was identified as the dominant positive ion when helium was used as the plasma supporting gas. This ionic species was believed to both serve as an energy carrier from the discharge into the afterglow, and to serve as a charge transfer reagent between He2+ and N2, leading to the formation of N2+. This ionic molecular nitrogen species is a key intermediate in the formation of protonated water clusters. 2.2.4. Chemical Sputtering/Ionization Techniques. This group of techniques is based on similar APCI-like ionization mechanisms as in DART, FAPA, etc. but with the specific use of desorption by charged solvent gaseous species formed by the plasma. Two techniques fall within this subcategory: desorption atmospheric pressure chemical ionization (DAPCI) and desorption corona beam ionization (DCBI). DAPCI was first described in the context of elucidating the DESI desorption mechanisms during the analysis of explosives.247 In DAPCI, gas-phase solvent vapors are ionized by corona discharge ionization. Charged solvent species thus
with a remote sampling backpack-wand platform, where sampling/ionization and mass analysis occur in the wand, and analyzer control, data acquisition and vacuum pumping units reside in the backpack.235 Sampling in the wand takes advantage of the “discontinuous atmospheric pressure interface” (DAPI) where a pinch valve coupled to a flexible inlet capillary periodically admits pulses of material from the atmospheric pressure region.236 An alternative to LTP MS for field in situ applications is the use of ion mobility analyzers. Jafari has recently demonstrated the applicability of LTP as an ionization source for DTIMS, with encouraging results for a variety of explosives.237 Precedent for this work can be found in earlier experiments by Franzke et al. who also investigated the use of a DBD for identical purposes.238 LTP (Figure 9) and DBDI make use of capacitively coupled plasmas that interact directly with the sample, and do not employ external heaters.157c The plasmas are generated by a DBD produced in mm-sized gaps. At atmospheric pressure, DBDs generate ‘‘cold’’ nonequilibrium plasmas. DBDs, also known as silent discharges, have been in use since the 19th century, and employ electrodes that are separated from the plasma itself.239 Typical dielectrics used to separate or coat the electrodes in DBDs include glass, ceramics and various polymers. The electric field in the DBD gap has to be high enough to produce gas breakdown even in the presence of this dielectric barrier. Because the above-mentioned dielectrics are less efficient at limiting the current at high frequencies (5−10 MHz), DBDs are generally operated in the AC mode. The application of DBDs and other ambient techniques to reaction monitoring has been recently reviewed by Zhang et al.1a Cooks, Ouyang et al. recently reported the use of LTP for the rapid detection of 13 explosives and explosives-related compounds with remarkable sensitivity in negative ion mode, reaching detection limits in the low pg range.240 A “reactive” LTP mode was also described, where trifluoroacetic acid (TFA) was added to the discharge support gas through a T-junction. An appealing aspect of this approach is that LTP, when compared with DESI for example, does not require solvents for operation. A miniature LTP probe that could be operated on a portable gas tank was the basis for a rapid in situ method for detecting agrochemicals on the surface or in tissue of fruit using a portable mass spectrometer.241 With this device, diphenylamine (DPA) was detected directly from the skin of apples in the store, readily allowing the distinction between organic and nonorganic apples. LTP has also been employed to detect fatty acid ethyl esters (FAEE) directly from bacterial samples as a means of rapid identification. LTP mass spectra showed highly reproducible and characteristic patterns which were examined 2287
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produced are directed at the sample surface to induce “chemical sputtering” of adsorbed analytes by charge buildup on the surface, which facilitates ion desorption and transport toward the mass spectrometer inlet. Recent DAPCI applications include the study of volatiles in various kinds of Fructus schisandrae to distinguish geographical origin,248 and detection of sinapine, a bioactive chemical usually found in various seeds of Brassica plants, in radish taproot tissue.249 DCBI is a more recent technique250 that can be operated in modes that seem equivalent to temperature-ramped DART,181 low-current FAPA, and DAPCI. DCBI uses a helium plasma sustained in the corona regime. Typical settings involve a 10− 40 μA discharge current under a 3 kV potential difference. Because the discharge is not distal and because plasma species are not filtered as in DART SVP and DART 100 ion sources, a narrow corona beam emitting from the ion source is visible, facilitating the localization of the sampled area. Thermal mechanisms and chemical sputtering mechanisms are most likely responsible for desorption. Solvent vapors can be selectively added to the DCBI probe, achieving DAPCI-like desorption and ionization. DCBI has been recently coupled to frontal elution paper chromatography for the rapid analysis of chlorphenamine in herbal medicines and dietary supplements,251 and used for the semiquantitation of illicit ingredients in weight-loss supplements.252
desorption and ionization events in time and space enhances ionization efficiency by reducing charge competition and ion suppression phenomena that occur when analyzing complex samples. In addition, ND-EESI provides the ability to perform real time, in vivo, rapid analysis of surfaces and biological specimens. However, it has the limitation that it is mostly useful for low molecular weight compounds, likely due to the difficulty in desorbing larger molecules with an unheated gas stream. In addition to the flow rate and velocity of the gas stream, efficient desorption, capture and transport of neutrals is very much affected by the angles between the desorption gas stream and the opening of the transport tube with respect to the sampled surface. Developed by Gu et al., the geometry independent neutral desorption device (GIND) is an enclosure with fixed openings for containing the desorption gas stream, surface interrogation area and analyte collection.254 For characterization of liquid/viscous samples such as milk, urine, toothpaste, honey and reaction process aliquots, the desorption gas stream can be bubbled through or over the samples.255 GIND and similar sampling devices maximize the efficiency of sample collection and transport, minimize fluctuations in signal intensities caused by geometry-influenced processes, can be used together with remote sampling strategies and increase sampling safety while preventing contamination from/to nearby areas.256 By using dopants in the EESI solvent, ND-EESI provides the flexibility to enhance selectivity and sensitivity for trace detection of specific analytes in complex samples, such as toothpaste.257 Selective ion/molecule reactions in a “reactive” ND-EESI method were used for lowering limits of detection of diethylene glycol (DEG) below the minimum regulatory requirement (∼0.00002%, weight percent in toothpaste) while achieving high-sample throughput. Wu et al. extensively used ND-EESI in negative ion mode to analyze cheeses for quality control purposes.258 Gas flow rates above 1 L min−1 were found to lower sensitivity by causing dilution of the neutral plume and shortening the neutral contact time with the EESI plume. One of the greatest advantages of ND-EESI is its applicability in nonproximate analytical applications suitable for real time screening of surfaces, process monitoring, and sampling from harsh and difficult sampling environments. The use of ND-EESI to detect trace explosives on different kinds of surfaces (skin, metal, gloves, leather, clothing, paper and glass) at up to 10 m distances from the ESSI MS setup has been recently demonstrated.254 However, optimization of desorption gas flow with increasing sampling distance is needed for maximum detectability. 2.3.2. Beta Electron-Assisted Direct Chemical Ionization (BADCI). Ionization of neutrals by β particles emitted from radioactive ion sources such as 63Ni foils is one of the most established methods of producing ions. Radioactive ion sources are very simple and relatively inexpensive to use. Although these ionization sources provide a stable and reliable production of ions, their use is restricted by a variety of safety, environmental and regulatory concerns due to their radioactive nature. The BADCI ion source addresses these concerns as it employs a simple, low-activity 63Ni element (∼10 μCi), safe enough for direct open air ionization and handling.259 The small activity of the BADCI allows its usage without extensive shielding while providing a large flux of primary ions. Neutral analytes are desorbed off surfaces into the ionization region by a heated gas stream. Similar to traditional 63Ni ion source usage, the BADCI probe can also be used to analyze volatile and semivolatile analytes. The BADCI microirradiator was prepared
2.3. Two-Step Thermal/Mechanical Desorption/Ablation (Non-Laser) Techniques
In contrast to methods such as DESI, DART, and AP-MALDI, where the desorption and ionization events occur concurrently, two-step ionization techniques function in two sequential steps: desorption/ablation of analytes by an independent method to create a plume of neutrals at or in the vicinity of the ion source followed by ionization to generate gas phase ions prior to mass analysis. Desorption of analytes in two-step nonlaser based techniques is achieved using a focused stream of gas (usually heated) or hot surfaces. Desorbed neutrals are either carried into the ionization region via a carrier gas or are generated in close proximity to the mass spectrometer so as to be entrapped into the vacuum drag. Neutral desorption extractive electrospray ionization (ND-EESI), beta electron-assisted direct chemical ionization (BADCI), atmospheric pressure thermal desorption-secondary ionization (AP-TD/SI), and probe electrospray ionization (PESI) are some of recently developed ionization techniques belonging to this family. 2.3.1. Neutral Desorption Extractive Electrospray Ionization (ND-EESI). In extractive electrospray ionization (EESI), introduced by Chen et al., neutrals from liquid sample spray are extracted and ionized by an ion swarm created by ESI of solvents.14a Uncharged analytes in the sample spray beam are ionized through charge transfer reactions taking place during collisions between neutral aerosols and ESI ions in the gas phase. ND-EESI refers to the setup where liquid, semisolid, and solid surfaces are impacted with a stream of gas to create a plume of desorbed neutrals that are subsequently delivered to the EESI ionization region.253 ND-EESI extends the use of EESI to extraction and ionization of analytes existing in solid and or highly viscous state. Neutrals are desorbed off liquid/ semisolid surfaces/bulk materials using a focused gas stream formed by flowing heated nitrogen gas through a narrow orifice (i.d. 1 to 0.1 mm). These desorbed neutrals are carried into a section of Teflon tubing by the desorption gas momentum and transported to the EESI ionization region. Separation of 2288
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resolution of ∼50 μm. The achieved spatial resolution was recently improved by the same research group by using a 30 nm heated atomic force microscope (AFM) probe tip.262 The AFM was coupled to the mass spectrometer with a vapor transfer line and ESI interface. The model compound caffeine was desorbed from a thin film on a glass surface achieving nanometer scale resolved surface spot sampling with mass spectral detection. This is a promising platform for automated surface sampling and analysis of systems in the submicrometer scale. 2.3.4. Probe Electrospray Ionization (PESI). Probe electrospray ionization (PESI) is a two-step technique involving a solid-sampling electrospray probe.263 The probe used in this technique is a needle (e.g., disposable acupuncture needles) which is inserted into a sample, picking up or coating the exterior with material for posterior analysis. When probing a biological sample, the small amount of water (∼ pL)264 carried on the needle surface together with the sample is sufficient to induce an electrospray when high voltage is applied to the probe. Since the ionization process proceeds through ESI-like mechanisms, intact molecular ions can be observed even from challenging organometallic compounds.265 This process can be automated by attaching the needle to a motorized controller to control the depth and rate of sampling. It is also worth mentioning that since the probe is a solid needle, there is no clogging, although one must be sure that the surface is clean if repeated measurements are to be performed with a single needle. The choice of needle material appears to be critical for some samples. For example, in high NaCl and phosphatebuffered solutions, the detection of gramicidin S was best on an etched tungsten needle, whereas myoglobin was detected at higher intensities with a titanium wire.266 More fundamental studies need to be conducted to explain these results, but this effect suggests that the physical properties of the needle267 (surface roughness, tip diameter, angle of tip, etc.), the surface tension of the solution, and the surface activity of the analyte need to be considered when applying PESI.268 The ability to quickly probe a sample with a small needle has been an effective tool to monitor hydrogen/deuterium exchange reactions.269 After 1 min of diffusive mixing of deuterium donor and gramicidin S solutions, sampling of the solution by PESI revealed the initiation of H/D exchange. Since the approach is rapid and consumes very little of the total solution, PESI sampling could be carried out for long periods of time (30 min or more) to monitor kinetics, as was done with cytochrome C at different solution pH values. Time-resolved mass spectra were acquired to observe the enzymolysis progression over 60 min. This study also utilized an auxiliary vapor generator placed orthogonal to the PESI probe and MS inlet to create an online dilution effect with organic solvent. This helps reducing the likelihood of corona discharge that may arise when sampling from aqueous solutions.270 An exciting subset of PESI applications is the intact analysis of biological tissues. Phytochemicals have been detected directly from tulip tissues, and the change in carbohydrate composition of the tulip bulbs monitored.271 Also, PESI can be used as a biological imaging probe.272 PESI created images of mouse brain focusing on the positive ion signals from lipids.273 PESI MSI allows for precise control over the depth and spatial resolution of the image, however the tissue is damaged which limits subsequent experiments to be performed on the same section. Although still in the initial stages of development, the potential biological niche of PESI could be in the analysis of intact proteins from tissue for potential top-down applications.
by electroplating a 10 μm-thick, 1 cm-long 63Ni layer with a total activity of 10 μCi onto the end of a 1 mm diameter, 5 cmlong copper wire. The ionization mechanism is expected to be identical to that observed in conventional radioactive and corona discharge ion sources. The β particles emitted by the radioactive element react with air to generate N2+ ions, which subsequently react with atmospheric traces of water through a series of ion−molecule reactions generating a series of charged clusters such as (H2O)nNH4+, (H2O)nNO+, and (H2O)nH+. When desorbed, neutrals interact with these charged clusters, undergoing charge exchange/transfer reactions to produce charged analyte species. Sample position, desorption gas flow rate and direction, and emitter position with respect to the mass spectrometer inlet require optimization for maximum sensitivity. The analytical performance of the BADCI probe was evaluated by analyzing common off the shelf drugs. Ibuprofen produced the active ingredient protonated and ammonium adducts at m/z 207 and 224, respectively; and a generic multivitamin produced the protonated molecules of niacinamide (m/z 123), pyridoxine (m/z 170), and biotin (m/z 245). Simplicity, stability, and ruggedness of the source make it very suitable as a portable and field operable device. 2.3.3. Atmospheric Pressure Thermal DesorptionSecondary Ionization (AP-TD/SI). Thermal desorption of analytes followed by secondary ionization has very often been used in MS and IMS experiments. Usually, a carrier gas or heated gas stream flows over the liquid or the solid sample placed in a heated sample holder/chamber/tube to desorb and transport analytes into the ionization region. In AP-TD/SI, the sample is placed in a glass test tube and rapidly heated in close proximity to a pneumatically assisted ESI source and the spectrometer inlet.260 In this setup the desorbed analyte vapors rise into the ESI spray path by convection and become entrapped and ionized by the ESI-generated ions. In one of the interesting applications of AP-TD/SI, bacterial spores were pyrolized at atmospheric pressure to detect the bacterial spore biomarker dipicolinic acid (DPA) as the dimethylated derivative (2Me-DPA) via in situ derivatization using tetramethyl-ammonium hydroxide (TMAH). At 180 °C, the protonated (m/z 168) and sodiated (m/z 190) DPA molecules were observed, and with heating above 200 °C thermal decomposition products such as protonated picolinic acid (m/z 124) and pyridine (m/z 80) were detected. For bacterial identification, pyrolysis products of the protonated (m/z 196) and sodiated (m/z 218) ions of dimethyl-2,6-dipicolinate (2MEDPA) was monitored. A calculated detection limit of 2MEDPA was near 0.1% by weight of spores. In another variation of the AP-TD/SI technique, Ovchinnikova et al. constructed a heated proximal probe through a creative and untraditional use of a MD 80 wand from a WD 1 soldering station.261 The probe had exchangeable tips (1.6 mm wide, 0.7 mm thick) and was digitally controlled for output temperatures in the 25−350 °C range at the probe tip. Positioned near the MS inlet and just above the TLC plates (no direct contact with sample on the plate) the probe was used to thermally desorb dyes, pharmaceuticals, explosives and pesticides separated on these substrates. Neutrals drawn into the ionization region were ionized by ESI or APCI. Later the same team modified the probe tips by machining the standard 250 μm diameter Weller LT1 soldering iron tips to a diameter of 50 μm. With the temperature held at 350 °C these tips were used for imaging surfaces at a scan speed of 100 μm s−1, probe-to-paper surface distance of 5 μm, and lane spacing of 10 μm, providing a spatial 2289
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PESI has been shown to detect proteins from detergent solutions,274 and therefore one could imagine using these solutions to wash tissue before PESI analysis to enrich difficult to image proteins (e.g., membrane and membrane-associated proteins). 2.4. Two-Step Laser-Based Desorption Ablation Techniques
2.4.1. Laser-Based Hybrid Techniques Coupled to ESI or Plasma Ionization. Overview. The coupling of laser desorption/ablation to electrospray or plasma secondary ionization techniques led to the development of a subfamily of laser based ambient two-step techniques (Chart 4). UV or IR
Figure 10. Schematic depicting a laser ablation electrospray ionization (LAESI) platform together with a fast imaging system (C, capillary; SP, syringe pump; HV, high-voltage power supply; L-N2, nitrogen laser; M, mirrors; FL, focusing lenses; CV, cuvette; CCD, CCD camera with short-distance microscope; CE, counter electrode; OSC, digital oscilloscope; SH, sample holder; L-Er:YAG, Er:YAG laser; MS, mass spectrometer; PC-1 to PC-3, personal computers). The cone-jet regime is maintained through monitoring the spray current on CE and adjusting the spray parameters. Black dots represent the droplets formed by the electrospray. Their interaction with the particulates and neutrals (red dots) emerging from the laser ablation produces some fused particles (green dots) that are thought to be the basis of the LAESI signal. Reprinted with permission from ref 275. Copyright 2007 American Chemical Society.
Chart 4 Two-step laser-based techniques.
lasers are used to ablate or desorb material from a surface, generating a plume that is merged with an electrospray ion plume from an electrospray source or with a reactive plasma beam from a plasma source. In this way, desorption and ionization processes are separated in space and time and can be optimized independently. Of particular note is that the coupling to an electrospray source allows the production of multiply charged ions, which is advantageous for the analysis of biological targets. A laser coupled to an ESI source was first used for ambient analysis in 2005 using a 337 nm nanosecond pulsed nitrogen laser in a technique called electrospray-assisted laser desorption ionization (ELDI).4 ELDI is still utilized; however mid-IR laser desorption/ablation has been more extensively used in the past few years. The most accepted name for this, now commercially available, IR laser-sampling/ESI ionization hybrid technique has been laser ablation electrospray ionization (LAESI).275 In LAESI (Figure 10), the IR-ablated sample is picked up by an electrospray plume, and the analytes are ionized in a fashion similar to conventional electrospray. In contrast, in infrared laser ablation metastable-induced chemical ionization (IRLAMICI) (Figure 11) the analytes are ionized by a plume of metastables through proton transfer reactions.276 The latter has been recently developed and described.1c,276 Briefly, IRLAMICI is similar to laser/ablation-FAPA277 and to laser desorption atmospheric pressure chemical ionization (LDAPCI),278 with the difference that measurements are performed in the open air and that the electrical discharge is operated in the glow regime, whereas LA-FAPA and LD-APCI operate in
Figure 11. Schematic of the IR-LAMICI ion source coupled to a quadrupole ion trap mass spectrometer. The inset shows the total ion current trace observed for the analysis of a Tylenol tablet with the laser turned off and on. Reprinted with permission from ref 276. Copyright 2010 American Chemical Society.
the glow-to-arc and corona regimes, respectively. Approaches similar to LAESI were alternatively reported as infrared laserassisted desorption electrospray ionization (IR LADESI),279 laser desorption electrospray ionization (LDESI),280 laser ablation mass spectrometry (LAMS),281 laser electrospray mass spectrometry (LEMS)282 or laser desorption spray postionization (LDSPI).283 Although these techniques are not necessarily identical to each other, the general commonalities in instrumental setup warrant their grouping together with ELDI and LAESI. In addition, when the laser is used to excite an exogenous matrix cocrystallized with the analyte using either IR or UV laser wavelengths, and a voltage (∼500 V) is applied to a stainless steel target plate, the technique is called matrix-assisted laser desorption electrospray ionization (MALDESI).284 2290
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surface of E. japonicus leaves. The observed similarity in the calculated averages of the energy distribution at a given sampling cone potential between LAESI and ESI suggested that the detected ions did not gain appreciable internal energy in the ablation process, being these findings consistent with the mechanism previously described. That is, the recoil-pressureinduced material ejection in LAESI did not alter the internal energies of peptides, vitamin B12, and thermometer ions. In addition, neither the sample temperature nor the repetition rate of the irradiation laser altered the internal energy distributions. Recent Applications. Laser-based methods provide high spatial resolution in the lateral direction, and allow for depth profiling in the z-direction. Nanosecond laser-based techniques have achieved spatial resolution of 100−150 μm for ELDI,293 of 40−400 μm for atmospheric pressure (AP) IR-MALDI MS,294 and of 200−300 μm for LAESI295 together with 50 μm depth resolution in the z-direction. Recently, AP scanning microprobe MALDI has achieved a higher spatial resolution of 5−10 μm for tissue imaging.296 Chemical imaging and MS analysis of cell populations at atmospheric pressure has broad applicability in biomedical research as well as potential for clinical diagnostics.1d,285c,297 In this particular application, infrared lasers show great promise due to their ability to efficiently remove material, their sampling speed, and their minimum microscopic-scale damage to samples which makes them useful in the analysis of live specimens. Recently, Nemes and Vertes285c have summarized the advantages of each of these techniques for in vivo local analysis and in situ molecular tissue imaging paying special attention to lateral imaging, depth profiling, and three-dimensional MSI. LAESI and AP-IRMALDI were among the first ambient ion sources to use mid-IR wavelength irradiation for in vivo sampling of biological specimens for MSI purposes.275,294 LAESI was found to achieve 1−2 orders of magnitude higher ion yields than AP IRMALDI290 probably due to the more efficient postionization of the more abundant neutral molecules in the plume. LAESI offers the possibility of direct chemical mapping of biomedical specimens in their native states and, as exogenous matrix is not used, background interferences in the low m/z range are minimized. The ability of LAESI MS to combine twodimensional lateral imaging298 with depth profiling295 to achieve three-dimensional cross-sectional imaging275 was shown in studies of rat brain and plant tissues, respectively. MSI methods in microprobe mode used for mapping molecules in tissues are usually based on the use of a rectangular grid of sampling spots. This leads to the averaging of mass spectrometric signal from multiple cells leading to information loss regarding cell-to-cell variability. Single cell analysis of plant tissue metabolites by LAESI MS has been successfully achieved by focusing the laser pulse through an etched GeO2-based optical fiber tip299 resulting in ablation spots of approximately 30 μm in diameter. Cell-by-cell imaging was implemented through the detection of sodiated hexose (m/ z 203), sodiated disaccharide (m/z 365) and cyanidin (m/z 287), the latter being a secondary metabolite responsible for purple pigmentation in onion (Allium cepa bulbs).299a This epidermal tissue was used as a model system to determine the correlation between the colored cells and the detected metabolites without affecting adjacent cells during laser ablation. The combination of this technique with orthogonal projections to latent structures discriminant analysis (OPLSDA) successfully enabled the selection of a small number of metabolites that accounted for most of the variance between
As all of these techniques are relatively similar, different authors in the field have suggested merging them under only one acronym to avoid confusion, but this topic still remains an open question. The parameter settings that characterize each of these techniques include the source geometry (including laser incidence angle and ionization source), laser wavelength, laser pulse duration, pulse energy, repetition frequency and the use or not of a matrix (endogenous or exogenous). Most implementations involve an incident laser beam normal (90°) to the sample, but some experimental setups choose to have the laser at an incident angle of 45° with respect to the sample. This is likely due more to space limitations within a laboratory and the availability of focusing optics than differences in desorption fundamentals. IR lasers are usually tuned for 2940 nm with pulses of 5 ns duration at 2−20 Hz and pulse energy between 100 μJ and 2.5 mJ. Endogenous water in the sample can act as ionization matrix and facilitate desorption,285 since water content of tissues is >70% at atmospheric pressure. Among the different ambient hybrid techniques, LAESI is the one that has been recently commercialized and will perhaps become more integrated into laboratories. LAESI uses an IR nanosecond laser to resonantly couple with the O−H stretching water vibration mode that is inherently present in biological samples. During mid-IR ablation, the plume of material is ejected as a consequence of the O−H absorption, whereas UV ablation proceeds via electronic energy absorption. However, higher amounts of ejected material can be achieved using mid-IR rather than UV energy at low laser intensities and high absorption coefficients, since light penetration depth is higher in the mid-IR than in the UV spectral region.285c,286 The ablation process ejects microscopic volumes from the sample in the form of a plume275,287 that is composed of particles,288 and free molecules289 with low ionization yields.290 The mechanism that has received general acceptance in LAESI proposes that analytes are desorbed from the substrate by the laser in a plume that expands longitudinally upward, and undergoes ion− molecule reactions with charged ESI solvent, or coalesces with the electrospray droplet cloud and subsequently ionizes through charge exchange, solvent evaporation, and Coulombic explosion.1b,291 Analyte desorption occurs in a two-step process initiated by rapid surface evaporation and microscale phase explosion from the ablated spot due to the approach of the local temperature to the critical temperature (647 K for water) in the first 1 μs after irradiation,287a,c,d creating a plume of vapor and microscopic droplets. When the expansion of this plume is slowed down due to the opposing force exerted by the atmospheric pressure, it comes to a halt at ∼2 mm above the sample surface, and collapses back onto the target. A recoil pressure-induced material ejection takes place in the second phase of the ablation projecting material up to 30 mm above the surface. Nemes et al.292 have recently applied the survival yield method to estimate the internal energy of the ions generated with LAESI for aqueous solutions and tissue samples. The results were compared to ions produced by ESI to determine whether the high temperature and pressure conditions affect energy deposition into solutes during the ambient ablation process. The extent of molecular fragmentation was quantified by calculating the survival yield (SY) as SY = IM/(IM + IF) where IM and IF consisted of the signal intensities of molecular and fragment ions, respectively. Benzylpiridinium salts were used as thermometer compounds to estimate the internal energy deposition when deposited and air-dried onto the upper 2291
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In a similar approach to IR LAMICI,276 the combination of a multiwavelength laser (355, 532, and 1064 nm) system with DART has been recently applied to the analysis of thin layer chromatography plates and was given the name of PAMLDI MS.186 The possibility of irradiating the samples with infrared, visible or UV energy provided multiple choices for the detection of different low molecular weight compounds according to their specific absorption wavelengths. The Nd:YAG laser was operated with a pulse duration of 10 ns, a repetition rate of 10 Hz, and an incidence angle of 45°. The DART source was operated with He at 2.3 L min−1 with the metastable gas stream parallel to the TLC plate so that the plasma reacted with the sample plume desorbed from the TLC surface. Protonated ions were mainly monitored during the analysis of dye mixtures, drug standards, and tea extract, which were separated on a normal phase silica gel with a detection level of 5 ng/mm2. Rhodamine B (m/z 443), Sudan III (m/z 353), and fluorescein (m/z 333) were analyzed in the positive mode with visible laser light (532 nm); whereas quinine (m/z 325), chloramphenicol (m/z 323), and gliclazide (m/z 324) were analyzed using UV laser light (355 nm); suggesting that photodesorption dominated the desorption process. Chinese tea was also analyzed in the positive mode using UV PAMLDI MS, and the identification of theanine (m/z 175), and caffeine (m/z 195) was successfully achieved.186 2.4.2. Laser Electrospray Mass Spectrometry (LEMS). A nonresonant femtosecond laser has been recently implemented to induce vaporization of nonvolatile samples, followed by ESI MS to postionize molecules prior to mass analysis.282 The advantage of using this type of laser is that no matrix is required, and that the sample does not have to be water-rich as nonresonant radiation absorption is enabled by the high intensity of the laser pulse (∼1013 W cm−2). A Ti:Sapphire oscillator seeds a regenerative amplifier to create a 2.5 mJ pulse centered at 800 nm with a duration of 70 fs, and a repetition frequency of 10 Hz. The incident laser beam is located at 45° with respect to the sample surface. Before impacting the sample, laser energy is reduced to 400 μJ pulse−1. The mass spectrum of monoolein vaporized using intense nonresonant fs laser pulses from an insulating dielectric surface demonstrated that thermal desorption was not required for transferring molecules into the gas phase. The [M − H2O + H]+ (m/z 339), [M + H]+ (m/z 357), [M + NH4]+ (m/z 374) and [M + Na]+ (m/z 379) ionic species were similarly observed from metal and glass substrates, suggesting that a resonant transition in the substrate, sample, or matrix is not required when an intense, nonresonant fs laser is employed.306 In the last years, this technique has been applied on a significant number of systems including biomolecules such as lysozyme, hemoglobin, ovalbumin, and phospholipids;307 organic explosives;308 inorganic-based explosive signatures detected through complexation with cationic ion-pairing agents;309 amphiphilic lipids and hydrophobic proteins adsorbed on metal and dielectric surfaces;306 trace amounts of narcotics and pharmaceuticals adsorbed on different surfaces;310 and plant tissue samples using compressive linear classification.311 As well, LEMS combined with multivariate statistical analysis was applied to discriminate between different phenotypes of Impatiens flower petals, demonstrating the ability to discover potential biomarkers from direct tissue analysis.312 The analysis of cytochrome c and lysozyme also suggested that femtosecond laser vaporization prevents thermal denaturation preserving the condensed phase conformation of the proteins upon transfer
epidermal cell populations of an Allium cepa bulb and a Citrus aurantium leaf and from human buccal epithelial cell populations.299a LAESI combined with OPLS-DA was also successfully applied to the study of oncovirus-infected cell lines, showing that phospholipids contribute to signal differences between the cell types in the spectrum region of m/z 660− 830.300 Specifically, phosphatidylcholines, and thymosin β4 (nominal mass = 4960 Da), were identified as potential metabolite and protein biomarkers as they were down-regulated in virus-infected cells. Antenna proteins and numerous tentative metabolites from intact cyanobacterial cells were also successfully annotated301 using LAESI. The coupling of IR LADESI to an atmospheric pressure standalone DTIMS has been recently developed and characterized for the first time.302 This LADESI DTIMS platform was applied to the detection of active pharmaceutical ingredients in antimalarial tablets collected in developing countries of SE Asia and the results were compared with ESI DTIM MS for validation purposes. A Nd:YAG driven optical parametric oscillator at 2940 nm with an incident angle of 90° with respect to the sample, a 20 Hz repetition frequency, and 2000 μJ pulse−1 energy was used. IR LADESI DTIMS analysis of intact pharmaceutical tablets was successful in the detection of piperaquine as [Pip + 2H]2+, chloroquine as both the singly and doubly protonated molecule and artesunate as the sodiated molecule, with reduced mobilities that differed by only ∼2% from ESI DTIM MS. The initial evaluation of this technique indicated that database of corrected reduced mobilities would be necessary for identifying different active pharmaceutical ingredients if this instrument were to be used as the core of a portable drug quality-testing platform. In another recent study, Fe-insulin and Cu-insulin complexes were detected during the analysis of insulin via laser desorption with ESI postionization (LDSPI) when copper or stainless steel sample plates were used, respectively.303 The target surface used in the analysis was proven to undergo interactions with the 1064 nm wavelength IR pulse laser of ca. 1.1 mJ pulse−1, leading to unexpected complexation reactions between the analyte and surface material. The authors concluded that gold or silver should be preferentially used to support the sample when metal−analyte complexation or adduction is not desired. In addition to the parameters related to the laser desorption/ ablation step, experimental setups for two-step laser ablation/ ESI techniques involve variables such as the stage height, the ESI-inlet distance, the sample-inlet distance, the plate voltage, and the ESI flow rate. These multiple parameters can dramatically impact the quality and intensity of the mass spectral signals. Optimization of this type of multiparameter system follows the “one-factor-at-a-time” approach, under the assumption that variables are independent from each other. In a different approach, fractional factorial design of experiments (DOE) was used to optimize the combination of parameters and settings that influence on the performance of IR MALDESI MS.304 The detection limit of small to medium sized proteins was successfully decreased by this approach by 4 orders of magnitude over previous parameter settings,305 using cytochrome c as model compound. The main factors that were found to be significant for IR MALDESI MS included the sample-to-mass spectrometer inlet distance, the stage height, the flow rate, and the laser fluence; being the sample-to-inlet distance the most influential.304 The ablation of a spot closer to the ESI emitter generated the greatest ion signal probably due to the greater area for interaction of the ESI and laser plumes. 2292
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Chemical Reviews
Review
ions, and [M − OH]+ (m/z 369.35) ions. The limit of detection for verapamil [M + H]+ (m/z 455.3) was 0.25 μM, similar to what had been reported for LAESI,275 and the estimated absolute LOD (170 fmol) was similar to what was estimated for acetaminophen in IR-LAMICI.276 Cholesterol and triglycerides from rat brain were ionized more efficiently by LAAPPI than by LAESI,298 but phospholipids were not successfully detected with this technique. This new approach produced ionic species that have not been previously observed in LAESI or IR-LAMICI, and has great potential for the analysis of tissue samples. 2.4.4. Laser Ablation Sample Transfer. Van Berkel and collaborators123 were the first to develop a combined atmospheric pressure surface sampling/ionization method based on the use of laser ablation in a transmission or reflection geometry to produce a sample plume that is transferred to a liquid droplet of 0.1 μL at the end of an autosampler syringe injection needle. The use of a transmission geometry provided spatial resolutions at 70 μm in diameter. Commercial ink samples containing rhodamine were used to test this sampling/ionization method. In addition, the capabilities of HPLC were efficiently introduced between the sample collection and the ionization steps to separate rhodamine B from rhodamine 6G.123 The Nd:YAG laser was operated at 532 nm with a pulse duration of 15 s and energy of 5 μJ pulse−1. No signal corresponding to the dyes was observed when the laser was not fired. The droplet capture approach was also replaced by a continuous flow LMJ-SSP, operated in a noncontact, surface sampling mode in order to capture, transport and ionize the material ablated from the surface providing additional means for MSI.124 The authors demonstrated the imaging capability of this method using ink lines and letters and inked fingerprints on microscope slides with approximately 100 μm spatial resolution using a 337 nm N2 laser with 11 ns pulse width, 10 Hz repetition frequency, and 60 μJ pulse−1. In a similar experimental setup, IR laser radiation was used to ablate biomolecules under ambient conditions that were then transferred to solvent droplets, which were either deposited on a MALDI target for off-line analysis or flow-injected into a nanoelectrospray ion source of an ion trap mass spectrometer.315 The authors evaluated the ability of mid-IR laser ablation with reflection geometry to transfer to solvent droplets angiotensin II, bovine insulin, cytochrome c proteins and complex biological samples such as human blood, whole milk and egg white to solvent droplets without inducing ion fragmentation. The laser was directed at the target at 45° angle and was tuned at a 2940 nm wavelength, with a 5 ns pulse duration, 2 Hz repetition rate, and a maximum fluence of 3 J cm−2. The optimum conditions for sampling consisted on having a large solvent droplet (2 mm diameter) suspended close to the sample (1 mm) so as to capture the largest quantity of material, with an efficiency estimated to be 1% (10 pmol in a typical experiment). In a different sample transfer approach, a mid-IR laser was used in transmission mode to ablate material from tissue sections under ambient conditions from a microscope slide for capture on a second slide, the latter acting as the target for vacuum MALDI MSI.316 The slide containing the sample was placed against the target slide with the sample side facing downward toward the latter with an adjusted gap in between. Indium tin oxide-coated microscope slides were used. The target slide was coated with a thin layer of nitrocellulose to
into the gas phase for capture and ionization in an electrospray plume at atmospheric pressure.313 The measured charge state distribution detected in vacuum was a signature of the condensed phase structure. The fast time scale of energy deposition for vaporization probably prevented thermal processes that would cause aqueous proteins to unfold. This work achieves an important goal for the field of MS that is the development of methods capable of measuring biomolecular structure in the condensed phase. 2.4.3. Laser Ablation Atmospheric Pressure Photoionization (LAAPPI). Vertes and co-workers314 have recently combined infrared laser ablation with photoionization (Figure 12), introducing a new ambient ionization technique for the
Figure 12. A schematic representation of the LAAPPI ion source (not to scale) with a photo of the ionization region shown in the inset. Reprinted with permission from ref 314. Copyright 2012 American Chemical Society.
analysis of compounds with different polarities. The mid-IR laser was operated normal to the surface (90°) with a 2940 nm wavelength to transfer energy to water molecules in the sample. The ablation plume was intercepted by an orthogonal hot solvent jet from a heated nebulizer microchip (∼240 °C) at ∼12 mm above the target. Toluene, anisole, methanol and their mixtures were used as spray solvents, atomized inside the microchip by means of a N2 gas flow and high temperature. This hot solvent jet allowed vaporization of the ablated sample droplets. The sample plume and the solvent spray were photoionized in the gas phase with 10.0 and 10.6 eV photons produced by a radiofrequency krypton discharge VUV photoionization lamp, positioned orthogonally with respect to the laser beam. The UV radiation ionizes solvents with low ionization energy (IE) (
