Ionization Mass Spectrometry ... - ACS Publications

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Ambient Sampling/Ionization Mass Spectrometry: Applications and Current Trends Glenn A. Harris, Asiri S. Galhena, and Facundo M. Fernandez* School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta Georgia 30332, United States

bS Supporting Information ’ CONTENTS Scope of this Review Spray and Solid
Liquid Extraction-Based Techniques DESI Fundamentals Instrumentation Applications EASI PESI ND-EESI and AP-TD/SI LMJ-SSP and LESA Plasma-Based Techniques DART Fundamentals Instrumentation Applications FAPA LTP and DBDI Microplasmas Chemical Sputtering/Ionization Techniques Multimode Techniques Laser Desorption/Ablation Techniques LAESI/IR-LDESI/MALDESI ELDI IR-LAMICI Acoustic Desorption Methods LIAD-ESI RADIO Other Techniques DAPPI SwiFerr BADCI REIMS Associated Content Author Information Biographies Acknowledgment References

4508 4511 4511 4511 4513 4515 4518 4518 4519 4520 4521 4521 4521 4522 4523 4526 4527 4528 4528 4528 4528 4529 4531 4531 4532 4532 4532 4533 4533 4534 4534 4534 4534 4534 4535 4535 4535

short) in the period ranging from January 2009 to January 2011. PubMed and Scifinder Scholar electronic database searches were performed independently by the authors. Full text articles were obtained and reviewed in all cases. 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”. The final collated list of references includes more than 290 papers. Analytical Chemistry’s current policy limits the number of references to a maximum of 250, focusing mostly on important trends and critical aspects. Therefore, we have chosen to include in the main text portion of this review only those references which we considered highly significant to the field. These were selected because of the originality of the application or the unique fundamental insights provided which could have implications to a multitude of applications. Additional references in the period covered by this review, but not discussed in the main text, are provided in the Supporting Information for completeness. Because of the rapid growth of the field of ambient MS, both in terms of number of techniques and their applications, we have chosen to provide a dual organization for the material covered. First, the main text groups techniques and their applications in the following categories: (1) spray and solid
liquid extraction-based techniques which involve electrospray ionization (ESI) or similar mechanisms, (2) direct and alternating current (dc and ac) plasma based techniques involving chemical ionization (CI) mechanisms, (3) plasma-based techniques where chemical sputtering-like desorption steps are followed by CI, (4) multimode techniques involving two of the principles discussed in previous sections, (5) laser desorption/ablation methods, (6) acoustic desorption methods, and (7) other techniques that do not fit into previous categories. Second, in addition to this “technique-centric” approach, we also provide a set of tables, which reference applications of ambient MS to the analysis of (a) environmental samples (Table 2), (b) food flavor and fragrances (Table 3), (c) forensics (Table 4), (d) homeland security (Table 5), (e) molecular imaging (Table 6), (f) pharmaceuticals (Table 7), (g) oil, polymers, and additives (Table 8), and (h) bioanalysis (clinical, metabolomics, proteomics etc.; Table 9). These tables do not include proof-of-principle papers or fundamental studies where only model analytes in clean matrixes were examined. In that sense, rather than being viewed as a comprehensive coverage of all recent

’ SCOPE OF THIS REVIEW

Special Issue: Fundamental and Applied Reviews in Analytical Chemistry

This biennial review discusses advances in the field of ambient sampling/ionization mass spectrometry (“ambient MS” for

Published: April 15, 2011

r 2011 American Chemical Society

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Table 1. List of Acronyms and Relevant References name

first report

AP-TD/SI

atmospheric pressure thermal desorption-secondary ionization

ref 101

N/A

BADCI

beta electron-assisted direct chemical ionization

ref 102

6

DAPCI

desorption atmospheric pressure chemical ionization

ref 176

2

DAPPI

desorption atmospheric pressure photo-ionization

ref 205

6

DART

direct analysis in real-time

ref 206

2

DBDI

dielectric barrier discharge ionization

ref 158

2

DCBI

desorption corona beam ionization

ref 180

2

DEMI DESI

desorption electrospray/metastable-induced ionization desorption electrospray ionization

ref 81 ref 27

3 1

DICE

desorption ionization by charge exchange

ref 80

1

EASI

easy ambient sonic-spray ionization

ref 207

1

ELDI

electrospray-assisted laser desorption ionization

ref 208

4

FAPA

flowing atmospheric pressure afterglow

ref 148

2

IR-LAMICI

infrared laser ablation metastable-induced chemical ionization

ref 193

4

LADESI

laser-assisted desorption electrospray ionization

ref 209

4

LAESI LDESI

laser ablation electrospray ionization mass spectrometry laser desorption electrospray ionization

ref 210 ref 23

4 4

acronym

Figure

LESA

liquid extraction surface analysis

ref 107

1

LIAD-ESI

laser-induced acoustic desorption-electrospray ionization

ref 17

5

LMJ-SSP

liquid micro junction-surface sampling probe

ref 211

1

LTP

low-temperature plasma probe

ref 159

2

MALDESI

matrix-assisted laser desorption electrospray ionization

ref 212

4

ND-EESI

neutral desorption extractive electrospray ionization

ref 213

1

PESI RADIO

probe electrospray ionization radio-frequency acoustic desorption and ionization

ref 214 ref 199

1 5

REIMS

rapid evaporative ionization mass spectrometry

ref 203

6

SwiFerr

switched ferroelectric plasma ionizer

ref 202

6

Table 2. Selected Environmental Application References technique

specific application

refs

AP-TD/APCI

herbicides from TLC plates

218

DAPPI DAPPI

PAHs in soil PAHs in urine and wastewater

200 201

DART

UV filters in water

122

DART

poplar pyrolysis products

124

DART

insoluble PAHs

139

DART

organometallic compounds

140

DART

sulfur-containing compounds in Chinese drywall

141

DCBI

pesticides in various types of water samples

181

DESI FAPA

chemical composition of atmospheric aerosols hydride-forming elements (As, Ge, Sb)

77
79 153

FAPA

herbicide mixtures by GC/MS

154

LTP

hydrogen peroxide in ambient air

167

nano-DESI

chemical composition of atmospheric aerosols

38

work in this area, this review should be viewed as the first biennial critical update to a series of previous review articles.1
11 To distinguish ambient ionization MS approaches from atmospheric pressure ionization techniques, we propose a set of basic traits that should be present in techniques to be included in the “ambient ionization/sampling” MS field. Ambient MS tech niques should enable (a) ionization in the absence of enclosures such as those typically found in ESI, atmospheric pressure

photoionization (APPI), atmospheric pressure chemical ionization (APCI), or AP-MALDI sources. This feature is critical when examining samples (“objects”) of unusual shape or size that could not be easily fit inside of an ion source enclosure or that would be critically disrupted or damaged when placed under vacuum. In other words, the technique should operate in the open air or ambient environment. (b) Ambient MS techniques allow direct ionization with minimum sample pretreatment such as preconcentration, extraction, derivatization, dissolution, or chromatographic or electrophoretic separation. Although this requirement can be relaxed to some extent in challenging applications of ambient MS, it is still one of the end goals of research in this field. (c) Should be interfaceable to most types of mass spectrometers fit with differentially pumped atmospheric pressure interfaces, without substantial modification to the ion transfer optics or vacuum interface. (d) Should generate ions softly, with amounts of internal energy deposited equal or lower than those in ESI, AP-MALDI, APPI, or APCI. The ambient MS field is now past its initial hype period and is finding its application niche, as will become evident in this review article. It is also becoming increasingly clear that, with some important exceptions, what is unique about ambient MS technology is not necessarily the ionization mechanisms themselves but the format in which the ion sources are designed/configured. Ambient MS techniques make use of well-established ionization principles such as ESI, CI, photoionization, etc., but in an open air direct ionization format which allows unique experiments to be performed on samples previously requiring significant sample 4509

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Table 3. Selected Food, Flavor, and Fragrance Application References technique

specific application

Table 4. Selected Forensic Application References technique

ref

specific application

reference 123

DART

cocaine and metabolites in human urine

DAPCI

melamine in milk

177

EASI

ink aging analysis

83

DAPCI DAPPI

differentiation of sea cucumber products fungicide detection on orange peels

179 200

EASI

counterfeit banknote analysis

84

ELDI

ink dyes on documents

191

DART

pesticides in fruits

119

LAESI/DESI

degradation of aged paper and books

183

DART

mycotoxins in cereals, grains and flours

131

LTP

works of art

172

DART

food-packaging additives

132

ND-EESI

diethyleneglycol in toothpaste

99

DART

melamine and cyanuric acid in powdered milk

133

DART

melamine in powdered milk

134

DART

release kinetics of taste-refreshing compounds in

135

DESI

chewing gum dried orange juice

34

DESI

artificial sweetener from Stevia leaves

74

DESI

agrochemicals on fruit peels and fruit/vegetable extracts 75

AP-TD/ESI

Bacillus spores for biotoxin detection

DESI

triglycerides in edible oils and margarine

76

DAPPI

illicit street-confiscated drugs

71

EASI

vegetable oils, butter, and lard

215,216

ELDI

lipids and casein in dried cow’s milk

21

DART DART

CWA thermal separation explosives

121 127

LTP

olive oil

163

DART

monitoring of adenine release for ricin activity assay 128

LTP LTP

agrochemicals on fruit peels and extracts melamine in milk

164 165,166

DART

CWA quantitation

129

DART

GHB detection in alcoholic and nonalcoholic

130

ND-EESI cheeses

97

PESI

92

human, cow and formula milk, bananas

preparation prior to MS analysis. Nondestructive surface analysis, spatially resolved analyte detection, multianalyte fingerprinting, matrixless imaging, and selective ionization enhancement by means of specific liquid or gas-phase chemical reactions are now possible with ambient MS, with most sample preparation needs effectively removed. While crafting this review article, we had to make some difficult choices to keep its focus tight and its length under control. Therefore, we have chosen to include in the Supporting Information short mini-reviews on three highly promising atmospheric pressure ionization techniques recently described: atmospheric pressure solids analysis probe (ASAP),12 laserspray,13 and paper spray,14 which we view as more closely related to APCI, AP-MALDI, and ESI, respectively, than to ambient sampling/ionization approaches. Also, this application review is unusual in the sense that two small subsections on fundamental aspects of desorption electrospray ionization (DESI) and direct analysis in real time (DART) are provided. We felt that because ambient techniques are still young, fundamental studies are still critically needed to understand the range of applications that can be enabled by their use. Some clear major trends were identified for the January 2009 to January 2011 period covered in this review. In terms of the number of publications, the top two techniques are DESI and DART with 32% and 27% of the publications, respectively. The third place is shared by publications involving low-temperature plasma ionization (LTP, 5%), easy sonic-spray ionization (EASI, 4%), and laser ablation electrospray ionization (LAESI, 4%). A total of 74 other papers involving more than 20 techniques account for the rest of the work in ambient MS in the last 2-year period. It is also interesting to look into the number of groups active in research involving the two most popular techniques, DESI and DART. In total, 34 groups published work involving DESI, with the Cooks group at

Table 5. Selected Homeland Security and Law Enforcement Application References technique

specific application

AP-TD/APCI explosives from TLC plates

ref 218 101

drink matrixes DESI

large area sampling of explosives

40

DESI

explosive detection with mini-MS

47

DESI LTP

organophosphorus CWAs with IM-MS explosives

48 160

LTP

explosives and related compounds

161

LTP

drugs of abuse

162

ND-EESI

explosives

98,100

Purdue University being the number one contributor with 29 papers, followed by the Chen group at Ohio University with 8 papers. Regarding DART, approximately 38 groups were actively involved in researching this technique, with our group and Cody et al. being the top contributors with 9 and 7 papers, respectively. Another interesting trend, in terms of applications, is that only a handful dealt with target molecules larger than 1 kDa, with an overwhelming focus on small molecule detection. Some notable exceptions include the detection of biomolecules from solutions with high concentration of salts using probe electrospray ionization (PESI),15 manipulation of protein charge states through continuous flow-extractive desorption electrospray ionization,16 the use of laser-induced acoustic desorption/electrospray ionization (LIAD-ESI) to detect proteins,17 the coupling of liquid sample desorption electrospray ionization (DESI) to a thin layer electrochemical cell to achieve oxidative formation and reductive cleavage of protein disulfide bonds,18 the detection of highmolecular weight proteins19 and heparin and heparin sulfate via DESI,20 protein detection via electrospray-assisted laser desorption ionization (ELDI),21,22 IR laser desorption electrospray ionization (IR-LDESI)23 and matrix-assisted laser desorption electrospray ionization (MALDESI),24,25 top-down protein sequencing using DESI,26 and the generation of multiply charged protein ions by radiofrequency acoustic desorption and ionization (RADIO).199 As expected, all these examples include some form of an ESI-like step for inducing ionization. No examples on 4510

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Table 6. Selected Imaging Application References technique

Table 7. Selected Pharmaceutical Application References

specific application

ref

technique

specific application

ref

AP-TD/APCI ink patterns

103

AP-TD/APCI

pharmaceuticals from TLC plates

DAPPI

65

DAPCI

tracing origin of pharmaceutical products

178

DAPPI

drug detection in urine and wastewater

201

phytochemicals in plant leaves, cholesterol in mouse brain tissue

218

DESI

human breast cancer

52

DESI

human arterial plaques

53

DART

antimalarial counterfeit drug detection

69

DESI

human lens lipids

54

DART

quantitation of drugs in biological matrixes

126

DESI

human brain tumors (astrocytomas)

55

DART

forensic illegal drug screening

142

DESI DESI

porcine and rabbit adrenal glands 56 cholesterol sulfate in human prostate cancer tissue 57

DART

preclinical pharmaceutical analysis for impurities, degradation products, isotopic

143

DESI

canine urinary bladder

58

DESI

human papillary renal cell carcinoma

59

DEMI

direct multimode detection of intact drugs

81

DESI

antifungal defense chemicals on tropical seaweed

66

DESI

RPTLC IM-MS of pharmaceutical mixtures

49

DESI

bacterial secondary metabolites

68

DESI

DM-MS of antimalarials

50

DESI

pharmaceutical tablet imaging

69

DESI

counterfeit antimalarials

69

DESI

banknote ink

84

DESI

veterinary drugs and hormones in animal feed 72

DESI DESI

rat spinal cord tissues lipids in mouse brain tissue

61,62 60,65

DESI

quantitative pharmaceutical analysis in deproteinized plasma

DESI

rat brain tissue

52,63,64

DESI

large surface area pharmaceutical detection

39,40

IR-LAMICI

pharmaceutical tablet imaging

193

DESI

drug detection in urine and wastewater

201

LAESI

lipid and small metabolites in rat brain tissues

182

LAESI

3D images of plant metabolites

187

DESI

SPME of illicit drugs and pain drugs in urine

217

LTP

depth-profiling of electronics coatings

171

EASI

drug detection off of molecularly

86

PESI

mouse brain tissue

92,96

after TLC

abundance and drug loading

after TLC

imprinted polymers

the ionization of large molecules by plasma-based techniques were reported.

’ SPRAY AND SOLID
LIQUID EXTRACTION-BASED TECHNIQUES The first widespread attention to the concept of ambient ionization/sampling prior to MS analysis was the introduction of DESI by Cooks and co-workers in 2004.27 DESI is an ambient ionization technique which combines features of electrospray and desorption ionization methods. In DESI, a high-velocity (90
200 m/s) pneumatically assisted electrospray jet is continuously directed toward the probed surface. This jet forms a micrometer-size thin solvent film on the sample, where rapid extraction and/or dissolution of analyte molecules occur. Liquid from this microfilm is dynamically dislodged by the incoming jet of fresh droplets, producing an ejection cone of analyte-containing secondary droplets which are sampled downstream by a mass spectrometer inlet. Formation of gas phase ions from secondary droplets is governed by mechanisms similar to those in ESI. DESI defines one of the major subsets of ambient MS techniques by its close relation to ESI in terms of involved instrumentation and ionization pathways, but with additional steps involved in the extraction/dissolution stages, and added analytical capabilities. The main benefit obtained from the use of spray-based ambient MS methods, like DESI, is their ability to sample molecules off of an intact sample surface via a liquid/solid extraction process without requiring time-consuming sample preparation as for ESI. Spray-based techniques share the benefits of improved throughput and the ability for spatially resolved molecular features to be probed under ambient conditions. The main differences within this category reside in the surface sampling approach used for desorption in each particular case. Figure 1 summarizes, in a

73

ELDI/MALDESI nonresonant femtosecond laser vaporization of vitamin B12

192

LESA

drug-like compounds from tissue sections

107,108

LIAD

TLC separation of drugs

198

LMJ-SSP

drugs detected from liquids, dried blood spots 106,107

SwiFerr

crushed over-the-counter tablets

202

TM-DESI

sulfur-containing drugs in plasma and urine

44,46

and mouse tissue sections

pictorial way, the main techniques that fall into the spray-based category defined by DESI and notes the basic steps involved in their sampling/ionization process. Within this section, we will detail the latest fundamental and application-driven research for DESI and all other spray and liquid sampling-based ambient techniques. DESI. Fundamentals. Although great strides have been made since the first DESI report in 2004, the fundamentals of the DESI desorption and ionization mechanisms are still being investigated to obtain a more complete picture of the prevailing mechanisms. In the timeline of this review, two particular fundamental topics have been probed: spray solvent composition and surface effects. Knowledge of how these two parameters affect DESI experiments results in a better understanding of the dynamics of the solvent droplets interaction with the sample/sampling substrate, leading to improved analytical figures of merits such as spatial resolution, sensitivity, and reproducibility. The erosion diameter of the sampled spot formed by the DESI spray jet has been thoroughly studied since this property is directly tied to the spatial resolution of the experiment. With DESI imaging MS being among one of the most soughtafter applications (reviewed below), surface properties (e.g., wettability) were looked into as a function of the percent of organic solvent in the spray liquid composition.28 Chiefly, for the 4511

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Table 8. Selected Polymers and Additives Application References technique

specific application

Table 9. Selected Bioanalysis Application References technique ref

specific application

ref

DART

mosquito metabolites

DART

phthalic acid ester additives in PVC toys

138

DART

ovarian cancer metabolome fingerprinting

145,146

DART DART

additives in PVC lid gaskets stabilizers in polypropylene samples

137 136

ELDI

rat brain tissue metabolites and multiply

21

EASI

nanofilm product coatings of flooring materials

87

FAPA

urine and yeast extracts

155

FAPA

polymer fingerprinting

151

LAESI

single cell metabolic profiling

188

TM-DART

insecticides in polymeric antimalarial bednets

45

LAESI

mouse brain tissue metabolites

186

LAESI LAESI

metabolic changes in virally transformed cells metabolites in the electric organ of

184 185

LAESI

multiply charged peptides and proteins for

organic solvents tested (ethanol, isopropanol, methanol, and acetonitrile), the erosion diameter decreased almost linearly with an increasing organic fraction in water. Optical microscopy revealed both glass and polytetrafluoroethylene (PTFE) surfaces sprayed with mostly organic solvent systems had a better defined elliptical sample spot, which was in contrast to a geometrically incongruous shape marked by dendritic extensions formed from the spray contact area in highly aqueous spray solvents. Additionally, it was observed that as analyte solubility increased, the spray erosion diameter decreased leading to an improved DESI efficiency (defined as the total ion count of the analyte per unit area sampled). In a related study on the use of nonpolar DESI solvents on an extensive list of 43 model analytes, the water/octanol partition coefficient describing the distribution of an analyte between an organic and aqueous phase was found to be a good indicator for the predicted DESI response in a particular solvent system.29 For instance, one class of molecules (thiamphenicol, dichloran, and furazolidone) partitioned more extensively in the aqueous phase and were detected only with 1:1 MeOH/H2O solutions with an exception of furazolidone, which was also observed in 1:1 CH3CN/CHCl3 solutions due to its slight solubility in chloroform. Molecules with preferential solubility in organic solvent systems (procymidone, simazine, trifluralin, bitertanol, 17-Rethynyl-estradiol, and Sudan dye I) were detected with both organic solvent systems CHCl3/THF (1:1) and CHCl3/ CH3CN (1:1) and not in 1:1 MeOH/H2O solutions, based mainly on their solubilities. A longer list of compounds showed substantial DESI response in both polar and nonpolar solvent systems, which was attributed to their solubility in both solvent types. Of note was a subset of para-substituted benzylpyridinium thermometer molecules that were tested to assess the relative internal energy deposition of organic-based solvent systems. Higher survival yields in organic solvents than aqueous solvents were observed, indicating a slightly lower degree of internal energy deposition. This phenomena parallels known ESI results and reinforces the droplet pickup mechanism proposed for DESI; however, a more rigorous analysis is required before a definite conclusion should be made. DESI solvent additives such as surfactants have also been studied recently.30 The key to using these additives was to select a surfactant that did not readily ionize in the polarity that the DESI analysis was used in, and to only use dilute (∼1 μM) amounts to prevent background ion interferences and to keep the instrument clean. Compared to the nonpolar solvent systems addressed in the previous paragraph, 1:1 MeOH/H2O solutions containing surfactants had lower detection limits and displayed a more stable signal. For instance, detection limits for challenging DESI molecules like melamine showed a greater than 3 orders of magnitude improvement with 1 μM surfactant-containing spray

144

charged proteins in solution

Torpedo californica 22

top-down proteomics LDESI

multiply charged peptides and proteins for

23

top-down proteomics LIAD-ESI

multiply charged peptides and proteins for

17

MALDESI

top-down proteomics multiply charged peptides and proteins for

24,25,190

top-down proteomics PESI

phytochemicals and carbohydrates in tulips

95

RADIO

multiply charged peptides and proteins

199

for top-down proteomics

solutions compared to standard 1:1 MeOH/H2O solutions (2 vs 4000 pg). It was believed that the addition of surfactant favorably altered droplet surface tension and sizes. When surfactantcontaining spray solutions were directly sprayed onto a surface, the spot areas were double the size (3.5 mm2 vs 1.7 mm2) of surfactant-free sprayed spots. For direct analysis purposes, an increase in the extent of surface sampling effectively translates into more analyte being sampled. Finally, increases to surfactant concentration (10 μM) resulted in faster surface sample depletion allowing for faster spectral acquisition rates. Research on various surface effects has been vital for the advancement of DESI applications. General guidelines for surfaces properties have been briefly outlined31 and can be summarized by noting that surface material composition, texture, and insulating properties are the primary factors to be concerned with. Nonconductive materials such as PTFE, polymethylmethacrylate (PMMA), and roughened glass share limited, if any, surface affinity for analytes thereby increasing the DESI desorption efficiency. Confocal microscopy of the surfaces revealed that three regions can be observed in the DESI-sampled area: an inner zone of eroded material, a perimeter of material that was formed from redistributed droplets, and an outer zone resulting from splashed droplets.32 Supporting these results were fluorescence and absorption microscopy studies of organic dye-coated glass surfaces after DESI analysis.33 The dye was rapidly removed from the elliptical impact region of the DESI spray jet, and the remaining material was redistributed onto the periphery of the impact region forming small rivulets of material. When all of the dye was visibly removed from the impact region by long exposure to the DESI spray jet, ion signal still persisted. It was hypothesized that the built up material in the rivulets may be the source of the material that was still being sampled from mechanistic pathways other than DESI. Although no direct evidence was given, the production of small Taylor cones at the ends of the rivulets may exist, but further research is required. These rivulets 4512

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Figure 1

were also observed when utilizing spray desorption material collection onto another surface followed by DESI analysis of the second surface.34 Increasing solvent flow rates of the primary spray desorption source increased the amount of collected material. However, not all material was removed from the sampled area as surface washing redistributed material away from the impact site to the periphery where rivulets were believed to form. This resulted in slow signal decay when the secondary surface was subject to prolonged DESI exposure. To directly probe the distribution of charge deposited on surfaces during DESI, a capacitive probe coupled to a metaloxide-semiconductor field-effect transistor (MOSFET) static charge detection circuit was used to image the distribution of surface charge on a PTFE surface.35 The highest charge density was localized to the small desorption area (∼1 mm2) formed by using higher gas pressures and smaller spray tip-to-surface distances which focused the spray jet. With the use of smaller spray angles, an increased area of charge distribution was observed which could improve large-area DESI analysis, later reviewed in the Instrumentation subsection. Of particular note was the effect of the inlet suction on the surface charge distribution on the surface. When the surface was placed in front of a MS vacuum inlet, two localized regions of charge density were formed, one directly in front of the DESI spray jet and one in front of the mass spectrometer inlet. Controlled experiments ruled out the effect of inlet voltages, so the presence of the two surface charge centers was believed to be derived from the pneumatic force of the DESI spray velocity and the other from the MS vacuum forces directly above the surface.

Instrumentation. Many groups have focused on the improvement and modification of the traditional DESI sprayer probe design to enhance the analytical figures of merit. In one case, a microfluidic nebulizer chip was made for DESI analysis of peptides to lower solvent flow rates and applied spray voltages.36 The DESI chips reached limits of detection an order of magnitude lower compared to a regular DESI spray probe for reserpine (0.5 vs 5 ng μL
1), bradykinin (0.75 vs 10 ng μL
1), and bovine serum albumin (1.25 vs 25 ng μL
1) due to the more focused gas and liquid jet targeting a smaller sample spot leading to more efficient desorption at low concentrations and more effective ionization at lower solvent flow rates. Along the same lines, Laskin and co-workers have developed a modified “DESI” source to sample small areas by using a self-aspirating nanospray capillary.37 The technique, named “nano-DESI”, utilizes a primary capillary to deliver a continuous low flow solvent stream to the surface while simultaneously forming a liquid bridge with a nanospray capillary (i.d. 50 μm) positioned close to the mass spectrometer inlet. This configuration separates the desorption and ionization steps which offers many potential benefits and is more closely related to the liquid microjunction surface sampling probe (LMJ-SSP) than to DESI, so a name change for this technique would be advised. One benefit from this approach is derived from improved sampling efficiency by allowing the user to more effectively sample a small area (diameter 100
300 μm) by adjusting the solvent flow rates without the worry of solvent washing away material. These benefits were shown to be important for the analysis of organic aerosols.38 A very stable signal was recorded with the “nano-DESI” probe for over 10 min 4513

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Analytical Chemistry indicating very low sample consumption, which is indicative that true DESI mechanisms are not in place. Given the size of the liquid junction (diameter 300 μm) and the surface concentration (∼0.14 μg mm
2), only 10 ng of the analyte (limonene) were consumed. Reversing aims, large-area DESI probes have been designed to meet industrial applications such as pharmaceutical cleaning validation.39 A large DESI sprayer was constructed similarly to a regular DESI probe but with larger diameter spray apertures and an interlocking plastic enclosure to cover the sampled area. The angle of the sprayer with respect to the surface was 40° from vertical which allowed a larger surface area (∼3 cm2) to be sampled. An ion transport tube up to 1 m in length was attached to the enclosure for nonproximate analysis of codeine applied to a stainless steel surface with a limit of detection of 10 ng cm
2, which exceeded the minimum industry standards by 2 orders of magnitude. This detection scheme was also demonstrated for octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocane (HMX) and cyclotrimethylenetrinitramine (RDX) when large areas (2000 cm2) of foam-core board were sampled with wipes (∼2.3 cm2) similar to airport security screening detection procedures.40 Detection limits as low as with 100 ng of explosive per 1.44  104 cm2 and 1.39  104 cm2 were found for RDX and HMX, respectively. With future optimization of this type of DESI probe and the advancement of mass spectrometer miniaturization, these results hold promise for homeland security applications. Barbula et al. used a novel rotating sample platform coupled to Hadamard transform (HT) time-of-flight (TOF) MS to improve DESI sampling rates.41 HT-TOF-MS removes the duty cycle constraints of typical TOF mass spectrometers by creating a 100% duty cycle measurement from a continuous ion beam to allow for maximum temporal resolution and sampling rates following DESI surface analysis. A patterned disk containing blank and chemically labeled regions was rotated in the DESI spray jet, and after data acquisition, individual regions were resolved at a rate of 50 samples s
1. When alternating labeled regions were marked on the disk, a maximum resolved rate of 80 samples s
1 was reported. It should be noted that these sampling rates could be difficult to achieve in a practical analytical laboratory with conventional scanning/trapping mass spectrometers, and sample placement on circular substrates would be challenging. New implementations of DESI have shown some improvements in ionization efficiency by using a high-voltage needle and grounded conductive surface decoupled from the nebulizing sprayer in an arrangement called electrode-assisted DESI (EADESI).42 EADESI uses a high-voltage needle placed orthogonally to a grounded surface. The solvent jet originates from a nebulizer adjacent to the needle at 40
45°. The uncharged spray jet desorbs material from the surface, and the secondary droplets are charged by the high-voltage needle. The needle height from the surface can be adjusted between 50 and 300 μm, depending on the applied voltage bias to prevent discharge with the sample plate. A low spatial resolution of ∼25 μm was reported with optimized conditions for rhodamine B (0.025 μM). A smaller spread of droplets onto the sample surface was said to be the major cause of the improvement of the resolution. It should be noted that samples must be directly applied to a metal or other highly conductive material for ionization. This will limit application to biological and other natural samples which, depending on their thickness and fluid content, may act as a dielectric on top of the conductive surface. EADESI also showed improve detection

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of larger molecular weight proteins (10
25 kDa) compared to DESI. Interestingly, the addition of a common MALDI matrix compound like sinapinic acid (SA) directly to samples improved signal intensity. Thorough follow-up studies should be pursued before conclusions are drawn. One of the parameters usually kept relatively constant in DESI is the incident sprayer angle to the surface (45
55°) and the surface to mass spectrometer inlet alignment. To make DESI analysis independent of these variables, a transmission-mode (TM) geometry was proposed to keep the incident spray and collection angles locked at 0°. The spray droplets pass through a porous sample like a mesh or screen where a liquid or dried sample can be deposited or sequestered. In this geometry, the only physical variables in the setup are the distances between the sprayer, the screen, and the inlet. The TM-DESI mesh properties must be carefully chosen since changes in material, strand size, open spaces, and surface functionality could influence results.43,44 Substrates with open spaces smaller than ∼150 μm allow for the least amount of scattering of the electrospray plume and favored transmission.43 This relates to using strands with smaller diameters (∼100 μm), since larger strand diameters have more total surface area exposed which scatter the plume and facilitate solvent and analyte spreading. Furthermore, it was shown that selection of the solvent composition and the mesh material is crucial. If the analyte favored partitioning in the solvent more than resting on the surface, desorption was rapid and ionization followed traditional ESI pathways. If the affinity of the analyte to the surface was greater, desorption was slower. Polypropylene, ethylene tetrafluoroethylene, and polyetheretherketone had better desorption properties than polyethylene terephthalate and nylon6,6 materials due to their porosity and their polarity and charging characteristics. Fluorescence microscopy experiments of meshes spotted with rhodamine 6G showed that the effective sample area was ∼0.8 mm2 with a symmetrical spray diameter of ∼1 mm under lower DESI solvent flow rates (5 μL min
1) at a sprayerto-mesh distance of 2 mm. Higher flow rates and greater distances resulted in larger and more irregular spray areas with diameters between 2 and 3 mm. Reactive TM-DESI was used to analyze insecticide-treated bed nets widely used in developing countries for malaria control by doping the spray solvent with ammonium acetate to promote cation adduction.45 A polyester bed net treated with deltamethrin (8 mg m
2) was analyzed by monitoring the ammonium adduct (m/z 523) to protonated molecule (m/z 506) to a fragment ion at m/z 281 transition. Additionally, the conditions used in this analysis permitted a spray area of 1 mm2, allowing for individual strands (diameter 250 um) to be interrogated since the open space (2 mm2) of the bed net was larger than the spray area. Sensitivity was improved by layering two to three layers of the mesh so that strands of the bottom meshes fell within the open spaces of the top mesh. This approach may aid quality control applications in assessing the uniformity of applied insecticide, but it does have a limit. Adding more layers may prevent the electrospray plume from passing through the mesh efficiently, lowering or completely eliminating the ion signal. The meshes and screens used in TM-DESI can be functionalized to improve affinity capture properties of the surface for selective screening applications.44 The so-called “surfaced-enhanced” TM-DESI applications have utilized capture agents with photocleavable bonds to facilitate mass-tagged detection of analytes. In one study, a highly selective and high-throughput 4514

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Analytical Chemistry method was used for capturing sulfhydryl (thiol)-containing analytes.44 The surface of a polyamide mesh (nylon 6,6) underwent an acid catalyzed hydrolysis to expose primary amine and carboxyl groups. Derivatization proceeded via binding of neutravidin with carbodiimide chemistry, introduction of spacer molecules (polylysine) to promote functionality and prevent steric hindrance, and completed with incorporation of a photolabile capture agent (e.g., VICATSH). Once functionalized, the mesh can enter the sample solution, washed of matrix, undergo UV lamp exposure, and be tested. For a simulated complex mixture containing salt and drug interferences, surface enhanced TM-DESI meshes were able to successfully capture and tag a thiol-containing molecule (captopril). Similar results in biological matrixes such as plasma and urine samples for thiolated drug compounds have also been shown.46 An interesting finding in this study was that relative signal intensity of two targeted analytes changed with molar concentrations. This effect was observed when molar ratios became large, as one component may act to suppress detection/sequestration of the lower concentration analyte. A potential remedy would be the addition of another capture agent to the mesh which expresses preferential binding to the lower concentration analyte to counteract this phenomenon. Some of the more exciting advancements with DESI implementation have been its coupling to miniature mass spectrometers and ion mobility techniques such as traveling wave ion mobility spectrometry (TWIMS) and differential mobility spectrometry (DMS). Negative mode DESI was coupled to a miniature MS for the detection of explosives.47 To cope with the excess solvent and gas that could interfere with the limited vacuum capabilities of the instrument, a discontinuous atmospheric pressure interface operating at a 1% duty cycle allowed ions to enter the ion trap where ion losses were minimal. Trace amounts of the explosives 2,4,6-trinitrotoluene (TNT), HMX, and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazone (Tetryl) were detected individually and in mixtures even in the presence of chemical interferences such as Windex. Detection limits ranged from 500 pg cm2 to 1 μg cm2, and large sample surfaces (75 cm2) could be analyzed in 22 s. With continued sensitivity and vacuum improvements of miniature MS instrumentation, routine field DESI analysis is in the not too distant future. DESI coupling to the Waters SYNAPT MS instrument has allowed for more facile ion mobility measurements to be made since the ion mobility cell resides inside the instrument under reduced pressures making ion transport easier.48 With utilization of the instrument’s Z-spray source, direct sampling off of solid phase microextraction (SPME) fibers was implemented for a series of organophosphorus chemical warfare agents (sarin, soman, tabun, cyclohexyl sarin, and VX). After optimization of instrument settings, DESI-TWIMS-MSn analysis was able to successfully identify each chemical by characteristic [M þ H]þ precursor and time-aligned parallel fragment ion (pseudo-MS3) species. Dacron swabs spiked with 5 μg of sarin were also probed with this system. Within 1 min, species in the TWIMS and MSn data were identified. A similar approach was also utilized for the detection of pharmaceuticals from nonbonded reversed-phase thin layer chromatography (RP-TLC) plates.49 Crushed cold and flu tablets were extracted in water, spotted onto the RP-TLC plates, and mounted inside the Z-spray source for DESI ionization. Ephedrine, paracetamol, and caffeine showed slight differences in ion mobilities which allowed more selective mass spectra to be extracted instead of using a summed mass spectrum.

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Differential mobility-filtering of DESI ions prior to TOF-MS detection was tested with several samples typically amenable to DESI analysis, including counterfeit pharmaceuticals and binary mixtures of isobaric chemicals of importance in the pharmaceutical and food industries.50 The orthogonal separation capability of DMS enabled filtering of unwanted chemical noise by preferentially selecting for ions within a particular differential mobility range. In DM-on mode, DESI-MS signal-to-noise ratios were improved by 70
190% when compared to DM-off mode. The chemical background prefiltering effect was highly encouraging, especially for future analysis of complex chemical mixtures and for imaging purposes. The ability to perform collision induced dissociation (CID) of preselected ions leaving the DM cell allowed for highly informative tandem MS-like fragmentation info to be obtained in a single-stage mass spectrometer. Finally, the performance of the DESI-DM-MS platform for the separation and detection of near-isobaric compounds was tested on an equimolar mixture of melamine and 5-hydroxymethylfurfural (5-HMF) deposited on a PTFE surface. Melamine has been commonly used to adulterate milk, whereas 5-HMF is formed as a result of thermal milk decomposition. In DM-off mode, the two species were marginally resolved in the TOF mass spectrum. However in DM-on mode, the isobaric melamine and 5-HMF ions were successfully baseline resolved and positively identified based on their appearance time in the mobility spectrum. Hybrid and multimode ionization sources have also been developed off the DESI platform. Solid and liquid samples on glass melting point tubes were introduced into a commercial ion source and tested in a hot gas stream for ASAP sampling or a heated ESI probe for DESI (see additional ASAP information in the Supporting Information).51 This approach takes advantages of the low volatility and higher molecular weight ionization capabilities of DESI with the ability for ASAP to ionize volatile and nonpolar species by switching between sources within a minute. In urine samples from a patient taking Levaquin, both ASAP and DESI showed the same protonated molecule of the protonated levofloxacin molecule (m/z 362.1514). Both techniques also shared similar protonated creatinine (m/z 114.0668) and protonated dimer urea ions (m/z 121.0726), but some differences were observed for other species. ASAP analysis revealed the protonated dimer of creatinine (m/z 227.1257) and a creatinine
levofloxacin complex ion at m/z 475.2099. DESI analysis produced the sodiated adduct ions in addition to the protonated molecules, which added improved identification capabilities. Applications. The MS imaging capabilities of DESI-MS have been one of the most rapidly advancing and attractive applications. As with all microprobe-mode MS imaging techniques, a mass spectrum is obtained at an individual pixel (sampled spot) and the sample is moved to the next adjacent pixel in a continuous fashion to construct ion images showing the intensities and spatial distribution of all or selected ions. DESI-MS imaging is advantageous over traditional imaging techniques because it requires no sample preparation such as histological staining and labeling for microscopy techniques, application of a matrix as in MALDI imaging, or the introduction of sample into high vacuum conditions as with secondary ion mass spectrometry (SIMS). Specificity for targeted analytes can be promoted through the addition of chemical additives in the spray solution for reactive-DESI imaging. However, there are limitations with DESI imaging. Routine DESI images have spatial resolution of 100
250 μm, which is larger than MALDI (g25 μm) and SIMS 4515

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Analytical Chemistry (100 nm),52 and ionization efficiency is limited to compounds that are typically ionized by ESI-like processes. Nevertheless, as this section will demonstrate, these limitations can be circumvented to gather rich data sets making DESI a powerful MS imaging tool. The ambient nature of DESI operation makes biological samples such as cryosectioned tissue samples amenable to direct analysis. The Cooks group has been the pioneer in this field, with specific attention given to the imaging of lipids in a variety of different tissue types such as human arterial plaques,53 lenses,54 liver astrocytomas,55 adrenal glands,56 prostate cancer tissue,57 and canine bladder carcinoma,58 which unfortunately cannot be fully reviewed due to space limitations. One particular class of lipids, phospholipids, which plays an important role in cellular function, signaling, and membrane composition in most all animal cells, has been particularly targeted by DESI practitioners. For these analytes and others described below, we expect that advances in multivariate statistical image analysis59 and improvements in three-dimensional visualization of tissue images60 will be powerful driving forces propelling future developments of DESI and other ambient imaging approaches. Positive and negative mode DESI analysis of rat spinal cord sections has received recent attention.61 In the outer white matter regions of the spinal cord containing myelinated axons, negative mode DESI analysis showed a homogeneous distribution of deprotonated plasm-glycerophosphoethanolamine (plasm-PE, 38:4) at m/z 750.6. In the same region but at a higher intensity were the glycerophosphoserine (PS, 36:1, m/z 788.5), glycerophosphoethanolamine (PE, 40:4, m/z 794.4), glycerophosphoglycerol (PG, 40:6, m/z 821.7), and glycerophosphoinositol (PI, 38:4, m/z 885.6) ionic species. In contrast, the gray matter which contains cellular bodies showed low intensity of the previous lipids but high abundances of deprotonated oleic acid (OA, 18:1, m/z 281.4), arachidonic acid (AA, 20:4, m/z 303.4), and docosahexaenoic acid (DA, 22:6, m/z 327.4). Positive mode DESI was able to ionize some of the lipids but in a much lower abundance. The [M þ Na]þ images of glycerophosphocholine (PC, 34:1, m/z 782.5) and sphingomyelin (SM, 42:1, m/z 837.8) were constructed, and these molecules were located in the white matter regions but in lower resolution images compared to negative DESI due to lower signal-to-noise ratios. The previous study laid the foundation for a follow-up study targeting the analysis of spinal cord lipids after traumatic injury.62 Specific differences were seen between the injured and normal spinal cord cross sections. For instance, the deprotonated AA (m/z 303.4) had a higher intensity in the injured dorsal (gray) region than in uninjured tissue. Other ions showing increased abundance in injured tissue were the deprotonated lyso-sulfatide (LysoST, 18:1) and diacylglycerol (DG, 32:1) at m/z 540.5 and 565.5, respectively. Overall, relative intensities of the detected fatty acids, DGs, and lysolipids were increased between 120% and 240% in injured tissues while there was a 30% reduction in the intensity of lipids at the center of injury and the surrounding areas indicating the hydrolysis of lipids in response to the spinal cord injury. Additional experiments utilizing reactive DESI with dinitrophenylhydrazine (DNPH) targeted malondialdehyde (MDA, oxidative stress biomarker) via the reaction between two DNPH molecules and the carbonyl groups of MDA. The detected ion was formed by dehydration of the two carbonyls of MDA and the amine group on two DNPH molecules to form the deprotonated [MDA 3 diDNPH
H]
ion at m/z 431.5. In

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lesions of the injured spinal cord, this biomarker was detected at twice the intensity as in healthy tissues. In these studies, isobaric species may be present in the untreated samples which interfere with the identification of different lipid classes. Therefore, high-resolution DESI analysis with Orbitrap MS was used to provide positive identification in a separate nonimaging experiment. This method required additional experimental steps such as resampling a tissue section which may decrease sample throughput. However, recent advances in coupling high-resolution MS (i.e., linear trap-Orbitrap63,64 and Fourier transform ion cyclotron resonance, FTICR65) to DESI imaging will eliminate these additional steps. In one case, rat brain tissue sections were analyzed on an linear trap Orbitrap mass spectrometer to improve selectivity in both positive and negative modes beyond unit mass resolution previously reported in the studies reviewed above.63 Many species differing by less than 0.1 u were imaged, including deprotonated diacyl-glycerophosphoserine (diacyl-GPSer, 40:6) at m/z 834.5284 (error, 0.5 ppm) and C20:0 sulfatide at m/z 834.5766 (error, 0.7 ppm) in negative mode, and the potassiated adducts of diacyl-glycerophosphocholines (diacyl-GPCho, 38:4) at m/z 848.5577 (error, 1.2 ppm) and cerebroside (C24:1 ClcCer) m/z 848.6386 (error, 1.1 ppm) in positive mode. Additionally, an automated DESI platform was coupled to a 9.4 T FTICR for high-resolution MS imaging.65 Modification of the commercial software package was required to synchronize the movement of the DESI stage, ion transfer optics, ion excitation inside the magnetic cell and the data acquisition to create ion images. Positive mode DESI analysis of mouse brain tissue revealed many isobaric species including baseline-resolved lipids at nominal m/z 832, which were identified as the protonated phosphocholines detected at m/z 832.5847 (exact mass 832.5851) and the sodiated adduct of the sphingolipid N-(15Z-tetracosenoyl)-1-β-glucosyl-sphing-4-enine detected at m/z 832.6630 (exact mass 832.6643). DESI imaging has not been limited to tissue sections, as it has also been applied to intact biological specimens. The potential for DESI in natural product discovery and secondary metabolite imaging has been demonstrated in two separate papers. Negative ion DESI-MS imaging has been shown to be a valuable tool for the analysis of surface-mediated antifungal compounds that serve as chemical defenses in tropical seaweeds.66 The seaweed species Callophycus serratus was found to secrete the antifungal compounds bromophycolide A/B only on lighter colored patches of the seaweed surface. These patches corresponded to damaged regions which can become susceptible to infection. The bromophycolides were shown to inhibit the growth of a marine fungal pathogen, Lindra thalassiae.67 In another example, DESI imaging was demonstrated in bacterial colonies. Bacterial secondary metabolites are often the target for pharmaceutical research, necessitating the need for new identification techniques. Imaging DESI analysis was also conducted to monitor the metabolic exchange between colonies of Bacillus subtilis 3610 (B. subtilis) and Streptomyces coelicolor A3(2) (S. coelicolor).68 Localization of the deprotonated antibiotic actinorhodin (m/z 629) was localized in the S. coelicolor colony when it was cultured with a laboratory domesticated strain of B. subtilis (PY79), a strain with impaired polyketide and nonribosomal peptide synthetase biosynthesis. This indicated a preferential growth advantage for the B. subtilis (PY79) strain by silencing antibiotic production in S. coelicolor, an effect not observed in wild type colonies. The pharmaceutical industry has benefited from DESI in both its high-throughput ability to screen intact medicines and 4516

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Analytical Chemistry through the direct screening of biological fluids. The detection of counterfeit drugs is of particular interest since it is a growing issue in developing countries and over the Internet. In one case, DESI was used to screen a collection of 14 antimalarial tablets representative of common pharmaceuticals available for purchase in Southeast Asia.69 Coupled with confirmation by both DART and two-dimensional diffusion-ordered nuclear magnetic resonance spectroscopy (2D DOSY 1H NMR), only 5 of the tablets contained the labeled active ingredient with the counterfeit drugs showing a mixture of wrong active ingredients (e.g., acetaminophen), wrong excipients (e.g., starch and sucrose), or a combination of both. Additionally, DESI images were constructed for mapping the surface composition of genuine and counterfeit pharmaceuticals allowing one to assess the homogeneity of the tablet. Reactive DESI has also been recently used as a routine tool in a countrywide drug quality survey aimed at obtaining the prevalence of poor quality oral artesunate antimalarials sold in the Lao PDR.70 Rapid screening of street-confiscated illicit drugs has also benefited from DESI-MS.71 Nine different samples revealed a range of illicit compounds such as the protonated molecules of amphetamine (m/z 136), MDMA (m/z 194), methamphetamine (m/z 150), heroin (m/z 370), and cocaine (m/z 304). Similar methods have been adapted to hormone and veterinary drug screening and forensics.72 For example, a drop of an illegal steroid cocktail was shown to contain a mix of the protonated molecules of methylboldenone (m/z 301.2188), progesterone (m/z 315.2342), and testosterone cypionate (m/z 413.3095) among many others. In two animal feed examples, DESI analysis showed some alarming findings. In one sample (Vytech 17HD), the labeled ingredients indicated were 17-halo-methyl-dianadrone, β-ecdysterone, and wild yam extract. However, in both MS and MS/MS scans, it was clear the sample was counterfeit since only the protonated molecule of caffeine (m/z 195.1) and its associated fragments (m/z 138.1 and 110.1) were observed. In another feed sample, the label claimed the sample contained a steroid that was “nondetectable” by other means called 17βmethoxytrenbolone. However, it was easily shown that the sample contained the anabolic steroid trenbolone (m/z 271.3) which was confirmed by NMR. These results highlighted the need for tighter regulation on prohormones and steroids in animal feed, which are easily obtained via online sources. Screening and quantitative analysis of drugs in urine and serum have been another potential pharmaceutical application. In one application, rapid (15 s) quantitative analysis of different drugs (amoxepine, atenolol, carbamazepine, clozapine, prazosin, propranolol, and verapamil) in plasma was completed after protein precipitation using acetonitrile.73 With precision and accuracy e 20%, limits of detection between 0.2
40 ng mL
1 were achieved. Quantitative analysis over the range of 20
7400 ng mL
1 showed linearity (R2 > 0.99). In a complementary study coupling DESI to solid phase microextraction (SPME) fibers for the detection of drugs in unprocessed urine, cocaine was quantified in the concentration range 20
1000 ng mL
1 (R2 = 0.9997) with a limit of detection of 25 ng mL
1. Food and agricultural DESI applications have grown in this time period too. A series of diterpene glycosides used as sucrose substitutes were qualitatively and semiquantitatively detected directly from small areas (g0.0225 cm2) of leaves from the Stevia shrub.74 Accurate mass DESI-MS and MS/MS monitoring revealed many glycosides including the chlorinated adducts of Rebaudioside C (C44H70O22) and F (C43H68O22) at m/z

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985.409 (error 3.7 ppm) and m/z 971.389 (error
0.6 ppm), respectively. Semiquantitative results were obtained by a standard addition method which consisted of spotting the surface of the leaf with a Rebaudioside D standard. A three-point calibration (R2 = 0.996) allowed for relative ratios of the glycosides to be determined based on their intensities to the standard. The approximate glycoside ratio in the leaf determined by DESI was ∼6% for Rebaudioside C and ∼4% for Rebaudioside F. Common agrochemicals such as pesticides, insecticides, herbicides, and fungicides were quantified from the surfaces of spiked fruit peels and extracts.75 A preliminary study was conducted comparing the DESI limits of detection for vegetable extracts spotted on PTFE substrates with direct infusion ESI-MS. Surprisingly, some compounds showed comparable or better limits of detection with DESI than ESI. For example, the pesticide ametryn had a limit of detection with ESI (1:1 MeOH/H2O) of 1.5 μg L
1. DESI analysis with the same solvent system on a PTFE surface resulted in a limit of detection of 30 μg L
1. However, with an optimized solvent system (80:20, ACN/H2O, 1% formic acid), the limit of detection was lowered to 2.7 μg L
1 for full MS and 0.1 μg L
1 for MS/MS. Furthermore, the same MS/MS experiment carried out in a high water content vegetable (tomato) matrix and the high acidic content fruit (orange) matrix resulted in limits of detection of 1.0 and 1.5 μg kg
1, respectively, highlighting DESI detection capabilities in challenging biological matrixes. More importantly, this study utilized isotopically labeled standards for quantitation purposes to improve the precision below 15%, which can be difficult for DESI. Citrus extracts with imazalil were used for quantitation and compared to LC
MS experiments. For three different citrus extracts (orange, lemon, and grapefruit), the DESI determined imazalil concentrations were comparable to LC
MS verified amounts: orange 0.19 μg g
1 (LC
MS) vs 0.16 μg g
1 (DESI), lemon 0.35 μg g
1 (LC
MS) vs 0.38 μg g
1 (DESI), and grapefruit 0.33 μg g
1 vs 0.30 μg g
1. Gerbig and Takats have shown the versatility of DESI for the analysis of triglycerides (TGs) in edible oils.76 TGs were detected as single and dimer ammonium adducts. In hazelnut oil, a common TG (52:2) species was detected as the ammonium adduct (m/z 876.7955), dimer ammonium adduct (m/z 1735.5551), and as in a complex with TG (54:3) (m/z 1761.5701), which was also detected as the ammonium adduct (m/z 902.8104) and dimer ammonium adduct (m/z 1787.5851). To aid in the identification of pine tree oil components, DESI analysis with and without the addition of ammonium was also pursued. When DESI of Sacha Inchi oil samples exposed to air and sunlight for 0, 5, and 8 days were tested, the increase in intensity of two particular ammonium adducts of TGs at m/z 868.7341 and 892.7320 were monitored to determine the extent of rancidification of the oil. After 5 days of exposure, the TG ammonium adducts with one, two, and three added oxygen molecules were observed at m/z 924.7304, 956.7196, and 988.7097. After the full 8 days of exposure, the sample solidified and the intensity of the oxidized species significantly increased. An emerging application of DESI has been the characterization of aerosols. Aerosols can be sampled from filters and quantified after standard calibration with solutions deposited on slides. For example, polycyclic aromatic hydrocarbons (PAHs)77 and organic acids (oxalic and oleic acids)78 have been quantified from biomass burning aerosols, with agreement to levels found by gas chromatography (GC)/MS. In another paper, the chemical aging of aerosols was investigated.79 This is 4517

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Analytical Chemistry typically very difficult to do with traditional ESI-MS due to the sample preparation steps involved, which introduce solvent
analyte reactions which complicate identification. When coupled to high-resolution MS, DESI analysis of limonene secondary organic aerosols revealed numerous oligomeric species in mass ranges (m/z > 300) that ESI-MS did not detect. The major cause of this difference was the limited interaction time of the incoming solvent jet in DESI (milliseconds) compared to total mixing in the ESI solvent stream. This led to the discovery that chemically aged organic aerosols contain conjugated nitrogen-containing species which may be responsible for light-absorbing properties of upper atmosphere aerosols. A technique, named desorption ionization by charge exchange (DICE) closely resembling reactive DESI, has been recently reported.80 DICE makes use of toluene molecular ions formed in the spray solution by electrochemical oxidation to ionize low polarity analytes by charge exchange that normally do not efficiently ionize with standard DESI spray solvent mixtures. Similar motivation has propelled the use of nonaqueous DESI spray solvents, as mentioned above,29 and the development of multimode ambient sampling ionization approaches, such as DEMI,81 discussed below. One interesting aspect of DICE is that due to the nonpolar nature of the spray solvent (toluene), it does not contain or pick up any appreciable amount of metal cations from the sample mixture, simplifying the complexity of the mass spectra obtained from biofluids, such as urine.82 EASI. EASI is a variant of sonic spray ionization, where the spray is directed at the sample surface, and previously referred to as desorption sonic spray ionization (DeSSI). The ion s ource itself is very simple and comprised solely of a Swagelok T-element, small ferrules, tubing, and a fused silica capillary at the exit. The other two entrances of the T are for the input of the solvent mixture (typically 1:1 MeOH/H2O with 0.1% formic acid) and gas (nitrogen or air at ∼200 psi). No voltage is required since the high-velocity sprayer forms small droplets, and ionization occurs due to a statistically imbalanced distribution of cations and anions in the spray. Other advantages of the technique include the lack of electrochemical or oxidation processes and the low charge density of the droplets, which may decrease solvent chemical noise. Desorption of analytes from the surface is believed to follow similar mechanisms as DESI. Many applications have been covered by EASI. For forensic and counterfeit studies, EASI has been used to identify inks,83 bank notes,84 and fabric softeners.85 With the study of inks, EASI was not only able to differentiate different types of ink based on the composition of dyes but also served as a “chemical clock” by showing degradation products of inks that had been aged in different sources of light.83 Ink fingerprinting was also key in a study assessing both DESI and EASI for the detection of counterfeit Brazilian, U.S., and European banknotes.84 Comparison of the two techniques in different laboratories, using different instruments and conditions, resulted in the same diagnostic ions, which were used to easily identify counterfeit bills based on the type of printing procedure. This demonstrated that the two techniques can be complementary to one another while providing robust, chemically rich information to be used alongside other counterfeit detection technologies for currency monitoring. In another study involving counterfeit products, EASI was used to identify the main surfactants used in fabric softeners.85 Low-quality and adulterated products were discernible by quaternary ammonium compounds containing only one

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fatty acid side chain instead of two. For example, in one adulterated sample the label listed a dialkyl imidazoline chloride as the surfactant, but the analysis revealed a monoalkyl surfactant (an amidopropyldimethylbenzylammonium salt). Surfaces modified with polymers have been analyzed by EASI in two different ways. In one study, molecularly imprinted polymers (MIPs) were made to improve the sensitivity and selectivity of EASI analysis by sequestering targeted analytes from complex matrixes.86 Phenothiazines in urine samples were selectively sequestered onto the MIP surfaces and remained after washing. EASI was able to desorb and ionize these molecules at a limit of quantitation of 1 μmol L
1 and at an analytical frequency of 6 samples h
1 (including all workup and analysis steps). Recoveries of the drugs ranged from 96 to 106%, and the MIP probes showed little variance (100 mM).15 A few fundamental articles have been published investigating the effects of the material15 and physical93 properties of the PESI needle. A comparison of etched tungsten, acupuncture (stainless steel), and titanium wire needles sampling “dirty” (high salt concentration) solutions demonstrated the importance of needle composition.15 In 150 mM NaCl and phosphate-buffered solutions, the tungsten needle provided a stronger signal for the detection of gramicidin S and the titanium wire for the detection of myoglobin. However, in urea solutions, all three needle types performed similarly. Although no rationale was given for this outcome, it is likely that both the hydrophobicity of the analyte, the emitter tip geometry, and needle surface roughness all play a significant role. Two of the drawbacks of PESI are the unknown amounts of sample picked up by the needle, the amount actually analyzed, and the need for water or other liquid to be present in highenough levels for ionization of the material. For the first problem, the sample could potentially be lost in the transition from sampling-to-ionization. An experiment was designed to study the volume of liquid picked up during a routine PESI experiment using a radioactive solution.93 A 35S-methionine radioactive (β-emitting) solution was probed by the PESI needle and then moved into a vial containing a liquid scintillation cocktail. The β-ray emission was recorded with a liquid scintillation counter. It was determined that between 0.35 ( 0.09 pL (mouse urine) and 5.69 ( 1.70 pL (mouse liver homogenate) adhered to the PESI probe. Liquid viscosity and surface tension were the major contributing factors for adhesion to the surface. Some biological tissues do not contain enough water or other fluids for effective ionization, or the sample may dry on the needle during its movement. A stream of water vapor in the ionization region has been shown to overcome this problem by allowing the vapor to condense on the needle surface, aiding in the dissolution of the fixed sample.94 Ionization occurs without affecting the stability or reproducibility greatly, but this approach does complicate the setup. With the use of the water vapor method, PESI was used to probe the parts of living tulip tissues.95 Different carbohydrates, amino acids, and phytochemicals were detected in vivo in various regions of the tulips. Specifically, the time-dependent carbohydrate content of the tulips during the first week of growth was investigated to gain insight into their metabolism. It was shown that within a week, the carbohydrate content decreased coinciding with the appearance and growth of shoots. MS imaging has been a target of recent PESI developments.92,96 In one study, mouse brain sections imaged at a lateral resolution ∼60 μm revealed many sodiated and potassiated PC ions in white and gray sections of the tissue. For example, [PC 36:1 þ Na]þ (m/z 810.6) and [PC 36:1 þ K]þ

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(m/z 826.6) were found predominantly in the white matter regions, whereas [PC 38:6 þ Na]þ (m/z 828.6) was found more in the gray sections. The galactosylceramide (GalCer) ions (e.g., [GalCer 22 h:0 þ Na]þ m/z 822.7 and GalCer 24 h:0 þ Na]þ m/z 850.7) were as abundant as PCs in the alveus of the hippocampus. Preliminary depth analysis of porcine retina was acquired showing changes in PC ion intensities with respect to incremental (20 μM) depth. Although these results are difficult to interpret since the different ionic intensity might be attributed to carryover from previous depths as the needle passes through the sample, they do offer the potential for future refinement of this method to analyze vertically inhomogeneous samples. Real-time reaction monitoring of protein denaturation, peptide hydrogen/deuterium exchange, and Schiff base formation have also been done by PESI.94 For these experiments, a 3 Hz driving frequency of the needle was used, allowing for a 0.33 s time resolution for the experiments. The driving frequency controls the speed of the needle moving toward the sample and to the mass spectrometer inlet. This approach may help to gain insight on reaction kinetics of unknown heterogeneous chemistries to better optimize reaction parameters. Additionally, since PESI has a high tolerance for salts, these types of reactions may be monitored in living biological systems. ND-EESI and AP-TD/SI. A general ND-EESI experiment is simple in concept and implementation. A room temperature (20 °C) nitrogen gas stream is flowed through a narrow opening (i.d. ∼0.1 mm) to form a sharp jet targeted to desorb analytes from only 2
3 mm above the surface. A low gas flow of 200 mL min
1 with gas speeds around 300 m s
1 is used to sample a small, targeted area (∼10 mm2).97 An optional enclosure, most commonly made of glass, can be incorporated to cover the sampling area and to ensure proper positioning of the gas jet and the sample transfer line. The sample transfer line then carries the neutral analyte plume to an extractive electrospray source. With separation of the desorption and ionization processes, ND-EESI is tolerant of complex matrixes and, when used with specialty sealed enclosures, it can increase sampling safety and prevent contamination from/to nearby areas.97,98 Matrix tolerance is due mostly to the uniqueness of the EESI process where charge-transfer collisions between the primary charged ESI solvent spray and a secondary neutral plume generated distal to the source induces analyte ionization. The neutral plume is delivered by the ND sampling transfer line. ND-EESI does have the limitation that it is mostly useful for compounds below m/z 300. This limitation is likely due to the difficulty in desorbing larger molecules with an unheated gas stream. Still ND-EESI has shown much promise for the investigation of biological samples in vivo with minimal ion suppression effects, based on the advances described below. Selective ion/molecule reactions in a reactive ND-EESI method were used for lowering limits of detection while affording high-sample throughput for the analysis of diethylene glycol (DEG) in toothpaste.99 DEG is toxic to humans and can build up within the body from repeated exposure (i.e., daily use of toothpaste). With the addition of ammonium acetate to the EESI electrospray solution, the two hydroxyl groups on the DEG molecule form NH4þ adduct ions (m/z 124) more readily than metal ion and proton adducts. Subsequent MS2 and MS3 assisted with identification via consecutive losses of NH3 (m/z 107) and H2O (m/z 89). After optimization of the experimental conditions, a limit of detection below the minimum regulatory requirement (∼0.000 02%, weight percent in toothpaste) with 4519

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Analytical Chemistry a relative standard deviation less than 8% and a recovery between 97.6 and 102.4% were obtained. Sample analysis could be completed in 2 s without memory effects as long as the DEG concentration in the toothpaste remained below 10% by weight. Zenobi and co-workers have extensively used ND-EESI to improve food trade and safety regulations.97 Cheeses from Switzerland were investigated in negative ion mode. High gas flow rates (>1 L min
1) were avoided since they negatively affected sensitivity by diluting the neutral plume and shortened the residence time of the neutrals in contact with the charged spray plume. Reproducible spectra were obtainable when sampling from regions of the surface with similar appearance. For example, slightly darker spots on the surface presented different spectra than other more homogeneous regions, indicating that chemical composition varied with surface quality. In total, 49 cheese samples were successfully differentiated by cheese type with principal component analysis (PCA). The data was insensitive to other physical attributes and pasteurization of the cow’s milk. Explosives have been investigated off of many different types of surfaces including human skin.98,100 In one study, a 4 m long sample transfer line was used to detect 10 pg of TNT as the radical anion at m/z 227.98 RDX, nitroglycerin (NG), and HMX were also detected in negative ion mode forming acetate anions [M þ CH3CO2]
at m/z 281, 286, and 355, respectively, when 1 mM ammonium acetate was added to the spray solvent. With positive ion conditions, triacetone triperoxide (TATP) was detected as the protonated molecule (m/z 223), ammonium adduct (m/z 240), and the sodiated adduct (m/z 245). The reported limit of detection on skin was 0.5
10 pg for most explosives, with a dynamic range covering 4 orders of magnitude. Of particular note was delayed testing of 1 ng of TNT and RDX on skin at intervals of 2
12 h after deposition. Because of the low volatility of the explosives, little sensitivity decay was observed and there was no correlation between skin temperature and signal intensity, demonstrating a desorption-based sampling mechanism. At a sampling distance of 10 m away from the mass spectrometer, another study showed complementary results with TNT, RDX, TATP, HMX, and NG using a geometry-independent ND source.100 Other experiments demonstrated the need to create independent calibration curves for specific surfaces (e.g., wet and oily skin). It was also observed that as the sampling transfer line increases in length (>3 m), signal response was delayed for a few seconds coinciding with a slow loss of signal intensity. Careful manipulation of the carrier gas rate mitigated these effects but at an expense of analyte dilution. Atmospheric pressure thermal desorption-secondary ionization (AP-TD/SI) is a variant of ND-EESI.101 As the name implies, analytes are thermally desorbed and introduced into the ionization region. Secondary ionization techniques such as APCI and ESI are commonly used in this implementation. In one study,101 liquids and solids were placed inside a glass tube wrapped in a heating wire and positioned in front of and below the ESI source. Thermally desorbed analytes entered the ESI plume and were detected by MS. The spores from B. subtilis were investigated by AP-TD/ESI for the detection of dipicolinic acid (DPA), a common bacterial spore biomarker used in homeland security applications.101 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) also appeared. Chemical background was reduced through in situ

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carboxylic group derivatization by methylation with tetramethylammonium hydroxide (TMAH). The protonated (m/z 196) and sodiated (m/z 218) ions of dimethyl-2,6-dipicolinate (2MEDPA) were seen and used in pyrolysis product monitoring for bacterial identification. A calculated detection limit of 2ME-DPA was near 0.1% by weight of spores. Another iteration of AP-TD/SI has used a proximal thermal probe to desorb analytes from TLC plates218 and for chemical imaging.103 A millimeter-sized pointed metal probe was placed close to or in contact with the sample surface. A soldering station was connected to the tip where the heating was controlled. The tips were made from machining traditional soldering tips (∼250 μm diameter) to a smaller size (50 μm diameter). Thermally desorbed species were introduced into the ionization region where they were ionized by ESI or APCI. Analysis of TLC plates coated with dyes, explosives, herbicides, pharmaceuticals, and other small molecules at a surface scan speed of 700 μm s
1 revealed that detection limits were correlated to the volatility of the analytes (TNT, 0.11 nmol; acetaminophen, 2.4 nmol; Sudan red 7B. 15 nmol).102 These probes were also tested for imaging of printed ink patterns at a scan speed of 100 μm s
1, achieving a 50 μm spatial resolution.103 LMJ-SSP and LESA. Ambient surface analysis can also be achieved via direct liquid extraction techniques. These probes extract analyte from the surface by contact of a continuously renewed liquid microjunction. The sampling probe is composed of two concentric tubes with the outer (larger) diameter tube delivering fresh solvent used for extraction and the inner tube suctioning the extracted analyte and residual solvent up through the probe. LMJ-SSP carries the analyte solution through the probe to an ionization source (most commonly ESI but APCI and laser-based techniques also possible). LMJ-SSP works best on hydrophobic surfaces that easily enable liquid microjunctions to be formed. When operating in a either a discrete single spot mode or a continuous scanning imaging mode, forming this microjunction can be difficult on wettable and/or absorbent surfaces. In these situations, aerosol application of silicone-based products onto the surfaces (e.g., HPTLC plates) can make LMJSSP possible.104 As with reactive DESI, LMJ-SSP can also utilize surface chemical reactions to improve desorption and ionization. In one example, electrochemically driven derivatization of thiol functionalities was achieved.105 The tagging of peptide and protein cysteine residues was facilitated by applying a small voltage difference between the sampling probe and the conductive surface via a battery powered circuit. The electrochemical oxidation of p-hydroquinone to benzoquinone provided sufficient agent to tag at least two cysteines on a peptide at an efficiency nearing 90% and flow rates of 5
10 μL min
1. To improve analysis speed, a LMJ-SSP was tuned to perform faster for high-throughput applications.106 Traditionally, the probe must be placed very close to the sample surface (∼20 μm) to form the appropriate LMJ. When sampling spot-to-spot, the probe must break contact and then reform the junction before starting again. with an increase of the distance to the surface (100
300 μm) and tuning the aspiration and liquid flow rates, a new junction could be formed rapidly. This was done by increasing the liquid flow-to-aspiration rate ratio so that excess liquid lowers from the probe tip to contact the surface. When the self-aspiration rate was increased, the LMJ would then sample the dissolved surface analytes. Sampling rates were around 30 s, sample carry-over was measured to be 0.1% for verapamil, and the dynamic range covered 2.5 orders of magnitude. These 4520

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Analytical Chemistry experiments were performed in solutions with the MALDI matrix R-cyano-4-hydroxycinnamic acid to demonstrate that LMJ-SSP coupled to ESI or APCI sources could perform complementary analysis of MALDI plates. Liquid microjunctions are not confined to in-house constructed probes but are commercially available via adaption of a robotic pipet tip system (Advion NanoMate chip-based infusion nanoESI system).107 This type of implementation is now called liquid extraction surface analysis (LESA).108 Spotted MALDI target plates,107 dried blood spots on paper,107 and thin cross sections of whole-body drug-dosed mice tissues107 have been studied with this technique. Another article investigated porcine ear tissue sections with the LESA system.108

’ PLASMA BASED TECHNIQUES Plasma based techniques comprise DART, flowing atmospheric-pressure afterglow (FAPA), LTP, dielectric barrier discharge ionization (DBDI), and microsplasmas (Figure 2). They involve the generation of a direct current or radiofrequency electrical discharge between a pair of electrodes in contact with a flowing gas such as nitrogen or helium, generating a stream of ionized molecules, radicals, excited state neutrals, and electrons. Some or all the plasma species are directed toward the sample, with optional secondary heating of the plasma gas stream to enhance desorption. Plasma ambient sampling/ionization techniques have the advantages of simple instrumentation, rugged construction, no need for solvents, and generation of singly charged analyte species that are more easily identifiable than for spray-based techniques, which also generate various adducts and multimers. Their use is mainly limited to analytes in a fairly reduced mass range, usually below 800
1000 Da. Differences between plasma techniques are sometimes not obvious, as not all experimental conditions are sufficiently well described to understand similarities and differences. The main differences usually reside in one of the following points: (a) if the plasma species are or not removed from the flowing gas (nitrogen, helium, or air) stream previous to interaction with the sample; (b) if the plasma gas stream is heated or not, and if the heating is performed by a supplementary heating element, or by Joule heating induced by the discharge current itself; (c) if the discharge is operated in dc, ac pulsed mode, etc.; (d) the regime in the current
voltage curve (i
V) where the discharge is operated; (e) if the discharge is in the point-to-plane or annular configuration; and (f) if the plasma is established within a macro (several millimeters or more) or micrometer-sized gap. Of the five techniques included in this subgroup, only DART is currently commercially available explaining its growing popularity. DART. Fundamentals. Because of several fundamental studies published in the peer-reviewed literature, DART, together with DESI, is one of the better understood ambient ionization/ sampling techniques. Three main fundamental topics of DART ionization have been investigated during the period covered by this review: (a) the fluid dynamic and thermal effects that prevail within the DART ionization region that result in changes in ion transmission,109 (b) the extent and pathways by which ions generated by DART can gain internal energy leading to unwanted in-source fragmentation,110
112 and (3) various models for ion generation mechanisms for both polar and nonpolar substances.113
116 Comparison of finite element computational simulations with matching experiments carried out by our group have demonstrated

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that gas velocity and temperature gradients play a central role in successfully transmitting ions generated within the DART ionization region into the atmospheric pressure interface of the mass spectrometer.109 It was observed that optimum sample placement was a fine balance between adequate and rapid sample heating to induce efficient thermal desorption and an abundant population of neutrals, and the losses that may occur when the gas trajectory lines are disturbed by the presence of a macroscopic sample placed between the DART ion source outlet and the mass spectrometer inlet. An important limitation of these simulations was that, due to computational limitations, suction from the mass spectrometer interface was not included in the models. However, the experimental results seem to qualitatively agree well with predictions from particle tracing simulations. It was also observed that due to heat losses to the environment, the effective gas temperature measured at different points within the ionization region was consistently lower than the DART gas temperature set through the software, an important point when considering the choice of experimental parameters, and for internal energy deposition studies described below. DART in-source fragmentation of labile molecules is a phenomenon that has been known for a number of years117 and has recently led to the discovery of unusual decay products of nucleotides and nucleosides.110 It is well known that metastable gas temperature has a critical role in the appearance of fragment ions. For example, systematic studies with the antifungal voriconazole demonstrated the increase in the relative abundance of fragment ions when DART gas temperatures were ramped from 50 to 500 °C.112 The presence of these fragments may make peak assignment difficult, including false negatives and positives. To further investigate these processes, our group recently completed a comprehensive study that used ion thermometry and computational fluid dynamics to attempt to identify the main sources of internal energy deposition in DART under standard operation conditions and compare the amount of internal energy deposited by this technique relative to benchmark softer techniques. The internal energy distributions of a series of p-substituted benzylpyridinium ions generated by both DART and ESI were compared using the “survival yield” method. DART mean internal energy values at glow discharge gas flow rates of 2, 4, and 6 L min
1 and at set temperatures of 175, 250, and 325 °C were in the 1.92
2.21 eV range. ESI mean internal energy at identical temperatures in aqueous and 50% methanol solutions ranged between 1.71 and 1.96 eV and 1.53
1.63 eV, respectively. The results indicated that ESI is a “softer” ionization technique than DART but that there was a certain degree of overlap between the two techniques for the particular TOF mass spectrometer used in this work. As a whole, when helium was used to sustain the glow discharge, there was an increase in DART internal energy with increasing gas temperatures and flow rates, indicating thermal ion activation and increased in-source activation within the first differentially pumped region of the mass spectrometer. There was no evidence of internal energy deposition pathways from metastable-stimulated desorption or excess energy released from large differences in proton affinities, but fragmentation induced by high-energy helium metastables was observed at the highest glow discharge gas flow rates and effective gas temperatures. In atmospheric pressure ion sources, ionization does not occur by a single, clearly defined mechanism as with vacuum techniques such as electron ionization (EI). For example, fundamental studies by Cody have shown that, although positive ions in DART are generally formed by proton transfer, molecular ions 4521

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Figure 2

can be produced as well by charge exchange mechanisms under very specific experimental conditions.114 DART shares some similarities with APPI in the sense that molecular ions are involved in the very first steps, which further react to produce charge or proton transfer products. The abundance of odd electron ions from a given analyte was found to correlate directly with the abundance of O2•þ, which was favored by small (3 mm or less) source-to-inlet distances, absence of excess moisture, and high (650 °C) set gas temperatures. Another interesting shared characteristic between DART and APPI is that dopants can be used to selectively enhance the production of odd electron species. For example, fluorobenzene, a typical APPI dopant with an ionization potential of 9.2 eV, can be added as a vapor during DART ionization of nonpolar analytes such as cholesterol. Under standard DART conditions (where O2•þ is not abundant), cholesterol predominantly forms the dehydration product [M
H2O þ H]þ. However, if fluorobenzene is added, a molecular ion is observed, in a similar fashion to that seen under conditions that maximize the abundance of O2•þ. Bartmess, Song et al. have published a series of interesting papers115,116 investigating positive and negative ion generation mechanisms in DART beyond those proposed by Cody. A “transient microenvironment” effect (TME) consisting of nine gas-phase reactions accompanied by the respective thermochemical data was proposed to address matrix effects in positive mode DART ionization. It was observed that mixtures containing 1 μg mL
1 naphthalene, 1,2,4,5-tetramethylbenzene, decanoic acid, 1-naphtol, anthracene, 1,3-dimethoxybenzene, 9-methylanthracene, 12-crown-4, N,N-dimethylaniline, and tributylamine in solvents such as methanol, toluene, hexane, and chloroform

produced very different DART mass spectra depending on the solvent chosen. The proposed nine-step TME mechanism suggests that analyte ionization, rather than proceeding by proton transfer from protonated water clusters, occurs by proton transfer from protonated microenvironment solvent clusters. Solvent molecular ions can also react with analyte molecules to produce both protonated analyte molecules and analyte molecular ions. This theory would also support the results described above regarding the use of fluorobenzene as a dopant, which would be part of the microenvironment of nascent DART ions. Negative ion formation mechanisms were also studied by Song et al., which compared DART with APPI, finding that the ionic products formed were similar, leading to four ionization mecha nisms including electron capture, dissociative electron capture, proton transfer, and anion attachment.116 Instrumentation. With recognition that sample introduction plays a critical role in DART, many reports presenting alternative ways of exposing analytes to the metastable stream have been published. An automated linear rail autosampler was used to map chemical plumes produced by accidental or intentional release of potentially toxic compounds using water-soaked cotton-swab wipe samples.118 Another swabbing technique, this time involving foam swabs, was recently developed to detect 132 multiclass pesticides on the surface of grapes, apples, and oranges entering the United States.119 In this application, a TM- DART ionization approach was coupled to high-resolution nontargeted mass analysis using an Orbitrap mass spectrometer, mitigating the loss in peak capacity caused by the lack of chromatographic separation. Detection was achieved consistently for 86% of the analytes at levels ranging 2
10 ng g
1. Partial separation was 4522

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Analytical Chemistry achieved by thermally ramping the DART gas temperature from 100 to 350 °C over 3 min. This thermal-dependent release of volatiles has also been employed in various DART applications such as detection of compounds from eucalyptus120 and to resolve ions produced by the chemical warfare agent sarin from isobaric interferences.121 TM-DART analysis was first implemented to detect and quantify insecticides directly from insecticide treated bed nets, showing great promise due to increased ion transmission and reproducibility, as suggested by fluid dynamic simulations.45 Because millions of insecticide treated bed nets are deployed worldwide for malaria prevention, detection of counterfeit products, and assessment of the remaining amount of insecticide after prolonged environmental exposure are two applications that can gain from the high-throughput characteristics of DART ionization. Transmission mode operation is highly advantageous in the case of bed net samples, as these can be “sandwiched” between two inert spacers and placed directly inbetween the ion source and the mass spectrometer inlet, allowing the DART plasma gas stream to interact with the sample in a flow-through fashion, therefore maximizing interaction with ionizing species. Direct DART desorption/ionization from solid-phase extraction materials has been reported in both stir bar122 and syringe formats.123 Both of these approaches are highly appealing as they remove some of the manual steps and potential elution losses involved in any preconcentration approach. DART coupled to stir bar sorptive microextraction was used to analyze environmental waters contaminated with UV filters. Semiquantitation was possible from polydimethylsiloxane-coated bars with repeatabilities in the 5
30% range and detection limits lower than 40 ng L
1. DART results were in good agreement with thermal desorption GC/MS, suggesting that this approach has the potential to be routinely used in environmental analysis. A modification to a DART ion source that incorporates a micropyrolyzer stage to study primary vapors and char products of poplar biomass in a stepwise fashion has been recently implemented.124 In this device, the stream of volatiles produced by the pyrolyzer was intersected with the DART metastable gas stream, enabling a direct decomposition/desorption/analysis experiment without sample preparation. In addition to DART-MS analysis, the sample surface was also monitored at various pyrolysis stages using Fourier transform infrared photoacoustic spectroscopy. The importance of this application cannot be underestimated, as the analysis of complex biomass is of critical relevance to address increasing demands in the energy and fuels field. DART has also been proposed as an ionization interface for LC
MS.125 Although most applications covered in this review are directed at avoiding any sort of chromatographic or electrophoretic separation, this work is still of potential importance. The coupling of LC to MS via DART was achieved via a simple interface consisting of a stainless steel or fused silica capillary directing the LC eluent into close proximity to the ion source exit grid electrode. It was found that DART was highly tolerant to phosphate buffer concentrations up to 120 mM in the mobile phase at typical flow rates of 1 mL min
1. Minimal contamination of the ion source or ion suppression were reported. Mixtures of parabenes and pyrazine derivatives were tested by LC
DART-MS with good linearity and limits of detection in the microgram per liter to milligram per liter range. One of the keys to the versatility of DART and other ambient ionization/sampling techniques has been the ability to be used on a variety of commercial mass spectrometers. DART was

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initially designed to work with cone/orifice atmospheric pressure inlets with no modification. However, many capillary inlet instruments could not handle the gas loads (especially of helium). As a result, a gas-ion separator tube assembly (GIST, also known as a Vapur tube) was designed to allow for the coupling of DART to these instruments. The GIST unit adds a small reduced-pressure region in front of the mass spectrometer capillary inlet to further reduce the vacuum load. The first demonstration of the GIST interface targeted improved quantitation of drugs in biological matrixes using automated sample delivery.126 A coefficient of variance of 3.1% was demonstrated with nine repeated injections of rat plasma containing 1 μM benzoylecgonine. In cases where the biological matrix suppressed the ion signal, as in the case of testosterone in raw rat plasma, a simple protein precipitation method was used to remove many of the interfering agents. Additionally, pharmacokinetic experiments involving the oral administration of 25 mg kg
1 of a proprietary compound to mice were used as a test bed for investigating differences observed between LC
MS/MS and DART-MS/MS results. Plasma samples taken over seven time points in three animals revealed the mean difference of concentrations between the methods ranged from 4.7 to 16.4%. With the exception of the Orbitrap,119 DART has been primarily coupled to mass analyzers of low to medium resolving power such as quadrupoles and TOF. Powell et al. described the first coupling of a custom-built DART ion source to two different (4.7 and 9.4 T) FTICR mass spectrometers. DART FTICR-MS was successfully applied to the detection with baseline resolution of mixtures of theophylline and diisopropyl methylphosphonate separated by only 27 mDa. Analysis of two simple food products (grapefruit flesh and havanero pepper) and a crude oil sample (NIST Heavy Sweet crude oil) demonstrated that the combined rapid screening capabilities of DART and the ultrahigh resolving power offered by FTICR-MS result in an extremely powerful tool for rapid characterization of complex mixtures. Applications. Similar to DESI, DART has been used for a multitude of applications due to both its ease of use, versatility, and commercial availability. The major driving force behind its widespread use was to develop alternatives to traditional GC and LC
MS methods. DART methods may not yield reproducibility as high as conventional GC and LC
MS, but the time and cost savings may ultimately balance out. Furthermore, DART accurate mass measurements can be rapidly obtained since external mass scale calibration is quasi-instantaneously performed through direct analysis of various calibrant compounds and because DART was initially preferentially marketed as a companion for orthogonal TOF-MS instrumentation. The most common DART calibrant in positive ion mode is diluted PEG 600, which provides good coverage over the typically acquired mass range (50
800 Da) and can be sampled easily after dipping a melting point capillary tube in the calibrant solution. DART has been identified in the homeland security field as a promising technique for surface and volatile analysis of explosives,127 biotoxins,128 and chemical warfare agents.129 For these applications, the ability to nondestructively sample from various surfaces and substrate materials allows for a quick first screening tool while more time-consuming but complementary investigations can be pursued in parallel. In an extensive study investigating the analysis of explosives (ethylene glycol dinitrate (EGDN), propylene glycol dinitrate (PGDN), Tetryl, pentaerythritol tetranitrate (PETN), mononitroglycerin (MNG), dinitroglycerin (DNG), trinitroglycerin (TNG), HMX, RDX, 4523

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Analytical Chemistry dinitrotoluene (DNT), amino-DNT, TNT, dinitrobenzene (DNB), trinitrobenzene (TNB), and picric acid) sampled from surfaces (glass, polyurethane foam, steel, wood, and asphalt shingle) and liquids (diesel fuel, creek water, and seawater), DART was shown to produce informative and simple spectra of the targeted analyte.127 The explosives were rubbed onto the substrates after deposition and drying on aluminum foil (60 kDa) enzyme, so direct analysis is not possible with DART. However, one can monitor the secondary processes that ricin invokes on eukaryotic cells, in particular, the release of adenine from the nucleic acid. Herring sperm DNA was used as a model substrate for ricin exposure, with uracil as an internal standard. Linearity for adenine was in the 2.9
740 μM range with the lowest concentration of adenine (2.9 μM) detected well above the limit of detection (SNR > 350). Timedependent ricin activity was observed for 50 h showing linearity in adenine production for the first 4 h at 53 ( 2 pmol adenine pmol
1 ricin h
1. Quantitative analysis of chemical warfare agents showed similar promising results.129 Linear calibration curves over 3 orders of magnitude for nerve agents (tabun, sarin, and VX) and a blister agent (sulfur mustard) were demonstrated. VX was detected as the protonated molecule and tabun and sarin as the ammonium adducts. Interestingly, the hydroxyl adduct cation was the base peak ion for sulfur mustard. This is an odd species to observe with DART. For confirmation of this species, negative mode analysis of sulfur mustard revealed the chlorinated adduct. For all compounds, linearity exceeded R2 > 0.99 and quantitative percent errors often averaging no worse than 3%. Food and beverage testing has seen its share of DART-related applications for both quality control and forensic purposes. γ-hydroxybutyric acid (GHB, “date rape drug”) was detected from 50 drink matrixes such as juices, sodas, wines, and liquors and proposed as a more reliable test than colorimetric assays.130 A lower limit of detection was set at 0.05 mg mL
1, which was lower than typically encountered GHB concentrations in real life samples (1.7
21.1 mg mL
1). Similar analytical merits were seen for fungal mycotoxins in cereals using either matrix-matched standards or 13C-labeled internal standards.131 A variety of mycotoxin standards (24) were tested in both positive and negative modes. Not all mycotoxins were detected (DON-3Glc, OTA, FB1, FB2, ergocornine, ergocristine, and ergosine), and it was believed that low volatility could be the cause since these compounds were heavier than other successfully analyzed mycotoxins (400
700 Da). Derivatization or alternative desorption methods could remedy this issue in future studies. Common anions and cations observed were the chlorinated (ADON, DON, Deepoxy-DON, FUS-X, NIV, ZEA, altenuene, alternariol, and alternariol-met) and deprotonated (ZEA, altenuene, alternariol, and alternariol-met) adducts and protonated (DAS,

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AFB1, AFB2, AFG1, AFG2, and sterigmatocystin) and ammonium (HT-2, T-2, and DAS) adducts, respectively. Comparative analysis of certified reference materials of DON or ZEA mycotoxins in maize flour, wheat flour, and ground millet by DARTMS and UPLC
MS showed that DART offered comparable results. For example, the mean amount of the mycotoxin DON in maize flour measured via external calibration and isotope dilution with DART was 459 and 486 μg kg
1 with relative standard deviations of 9.0 and 5.9%, respectively. Compared to the mean value (500 μg kg
1 maize flour) and relative standard deviation (4.1%) obtained by UPLC, the results obtained with DART are certainly similar. Food packaging was tested for additives like plasticizers, antioxidants, colorants, grease-proofers, and ultraviolet light stabilizers.132 Attention should be drawn to the detection of some high molecular weight (∼1000 Da) compounds in this study. For instance, a plasticizer (epoxidized soybean oil) and an antioxidant (Irganox 1010) were both detected as the protonated water adducts (m/z 992.8 and 1196, respectively). The epoxidized soybean oil contained a mixture of epoxidized triglycerides, diglycerides, and fatty acids with the base peak belonging to the epoxidized triglyceride of linoleic acid. The protonated water peak for Irganox 1010 was also accompanied with a less intense protonated molecule peak (m/z 1177.7). DART has also been applied to the detection of melamine contamination in milk powder. In one study, melamine and cyanuric acid were detected from powdered milk samples.133 Limits of detection for melamine and cyanuric acid were 170 and 450 μg kg
1, respectively. Isotopically labeled standards (13C3melamine and 13C3-cyanuric acid) were used for quantitation. Instant H/D exchange occurred with deuterated melamine analogues (melamine-d6). The limit of quantitation was 450 and 1200 μg kg
1 for melamine and cyanuric acid, respectively, with relative standard deviation in the 5
7% range for standard mixtures. In real life samples of dried milk, condensed milk, and dried cheeses, good agreement was seen among DART and LC
MS/MS methods with poorer results seen for an ELISA assay comparable to both MS metrics. The DART-MS and LC
MS/MS results for condensed milk analysis showed comparable mean values (4140 and 3958 μg kg
1) and relative standard deviations (6.2 and 2.8%). Differentiation between 5-hydroxymethylfurfural (5-HMF) and melamine can be difficult without tandem MS or highresolution MS capabilities since they share the same nominal m/z (127). In a clever experiment, Dane and Cody134 operated DART with argon as the discharge gas and acetylacetone and pyridine reagent “dopant” gases to selectively ionize melamine. The metastable energy states of argon (11.55 eV 3P2 and 11.72 3 P0) are lower than the ionization energy of water (12.6 eV); therefore, no Penning ionization of water occurred. Direct Penning ionization of either compound was not observed, so a multistep reaction sequence involving dopant gases introduced into the ionization region was used to selectively protonate melamine. First, metastable argon Penning-ionized acetylacetone. This molecular ion then reacted with a neutral acetylacetone molecule protonating it. The proton affinity of the acetylacetone molecule was lower than pyridine (873.5 vs 930 kJ mol
1), and pyridine thus captured the proton. Finally, the protonated pyridine transferred the proton to melamine. Although no literature values were found for the proton affinity of melamine and 5-HMF, these results demonstrate that melamine must have a proton affinity greater than pyridine, and both 4524

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Analytical Chemistry have affinities greater the 5-HMF. As noted earlier, this study utilized argon as the metastable gas and has been the first and only reference to date that uses this DART approach. A unique investigation of the release kinetics of a chewing gum flavor agent (WS-3, cyclohexanecarboxamide) in saliva was pursued with DART and compared to LC
-MS.135 A custom sample probe was made to reproducibly hold the sample in the gas stream. The probe consisted of a 0.12 mm diameter metal (nickel chromium) wire wrapped around a 0.80 mm  0.80 mm disposable syringe needle with a sealed hole. This probe could be rapidly made and reproducibly placed with the help of a custom assembly attached to the DART source. The sample probe provided a medium that conduced heat efficiently, provided surface “roughness” for the liquid to adhere, and was small so that the reactive gas dynamics were not severely disturbed. Compared to conventional melting point capillary probes, maximum signal intensity was higher for the new probe and the relative standard deviation was improved 3-fold over 4 decades of concentration range (0.1
1000 ng mL
1). In regards to the release kinetics of WS-3, four subjects gave 10 saliva samples over an hour of both free and encapsulated WS-3 in an extruded carbohydrate carrier. The levels of free WS-3 started low (1 μg g
1 saliva) and seemed to stay constant or gradually increase after 10 min of chewing (maximum level of 10 μg g
1 in saliva as shown in one subject). Conversely in encapsulated form, WS-3 levels spiked within the first 3 min to levels of 5
25 μg g
1 saliva. A sudden decrease in levels was then observed in the next few minutes until stabilization after 10 min near 3
10 μg g
1 in saliva. These results reflect the differences in saliva secretion between the subjects and the release of flavor quickly in encapsulated forms. Polymers and additives have been also investigated by DART since most low-molecular weight polymer subunits, side chains, and initiators contain functional groups that readily protonate. Additive stabilizing agents such as those from the Irganox, Irgafos, and Tinuvin types, among others, were investigated in both toluene extracts and directly from polypropylene polymer samples.136 For these stabilizers, common observed ions were the deprotonated anions and the protonated molecules. Positive mode ionization provided the best sensitivity with limits of detection below 1 mg L
1 except for 1 of the 21 tested samples (Tinuvin 328, 50 mg L
1). Intact polymer samples (0.5 cm  0.5 cm pieces) with multiple additives were all successfully investigated. For example, the additives Tinuvin 770, Tinuvin 234, Irgafos 38, and Chimassorb 81 at concentrations of 10 mg per 5 g of base polymer (polypropylene) were successfully detected as the protonated molecules at m/z 481.4017, 448.2373, 515.3637, and 327.1931, respectively. Additives in poly(vinyl chloride) lid gaskets were targeted for detection since their introduction into food products is regulated.137 Toluene extracts of gaskets were used to identify numerous compounds such as phthalates, fatty acid amides, tributyl O-acetylcitrate, dibutyl sebacate, bis(2-ethylhexyl) adipate, 1,2-diisononyl 1,2-cyclohexane-dicarboxylate, acetylated mono- and diacylglycerides, epoxidized soybean oil, and polyadipates. Similarly, phthalic acid esters were detected from poly(vinyl chloride) toys.138 Phthalic acid esters are under safety scrutiny for potential toxic effects in the health of children. Limits of detection for benzyl butyl phthalate, bis(2-ethylhexyl) phthalate, and diisononyl phthalate were 0.05%. Other additives such as dibutyl phthalate, di-n-octyl phthalate, and diisodecyl phthalate had a slightly higher limit of detection of 0.1%. Both studies were able to demonstrate that the detection of additives could be

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conducted from intact polymer samples or liquid extracts which required minimal time for preparation. Compared to traditional verification techniques which may exceed 16 h for preparation alone, complete analysis with DART took only 8 min. Environmental contaminants such as PAHs are insoluble and incompatible with many typical analysis methods. DART was able to quickly (3
5 s sample
1) screen for pure PAHs off of melting point capillary tubes.139 The protonated molecules of 1,2,5,6-tetrabromocorannulene (m/z 566.7236), diacenaphtho[1,2-a:10 ,20 -g]-corannulene (m/z 499.1484), diphenanthro[9,10-a:90 ,100 -g]-corannulene (m/z 551.1797), tetraindenocorannulene (m/z 547.1502), decachlorocorannulene (m/z 594.6904), 1,3,5,7,9-pentachlorocorannulene (m/z 421.8809), and peri-bis-dibenzo[a,g]corannulene (m/z 697.1947) were detected with mass accuracies better than 3 ppm. Protonated molecules and molecular ions were also detected from headspace vapor from organometallic compounds containing As, Fe, Hg, Pb, Se, and Sn.140 Additionally, protonated metal ions for some of the compounds were observed. For example, tetraethyllead sampled in helium showed the protonated lead ion (m/z 209) in addition to the molecular ion of ethyllead (m/z 237), protonated diethyllead (m/z 267), the molecular ion of triethyllead (m/z 295), and the water adduct of the protonated molecule (m/z 343). Sulfur containing species in drywall products were detected in negative ion mode.141 S2
(m/z 64), SO3
(m/z 80), and S3
(m/z 96), among others, were only detected from samples of drywall made in China. The high-throughput surface sampling qualities of DART have been exploited for forensic pharmaceutical drug screening. The detection of counterfeit antimalarials with DART has also been performed and briefly reviewed previously in the DESI Applications section.69 In a validation analysis of illegal drugs,142 limits of detection for target compounds (heroin, alprazolam, cocaine, testosterone propionate, and trazodone) were determined to be 0.05 mg mL
1. Then, 553 case specimens were tested by DART and compared to GC/MS. For identification/screening purposes, the results for all but one sample were identical. The one sample exception contained heroin and the unusual cutting additive yohimbine ([M þ H]þ, m/z 355.2001). Although this ion does not interfere with the detection of heroin ([M þ H]þ, m/z 370.1654) per se, the isotopic (13C) peak of an unidentified concomitant species at m/z 369.2147 positively interfered. The pharmaceutical industry has utilized DART in preclinical trials and for the quantitation of drugs in biological fluids. For example, photodegradation analysis of encapsulated photosensitive active ingredients was completed with DART.143 Although the active ingredient was not identified in the text by name, the corresponding protonated molecule and dimer were detected in the control (not exposed to UV light) and exposed in different capsule colors (white, orange, and green, increasing in UV protection). A degradation product was detected in UV-exposed capsules with relative increases in intensity corresponding to the lowest protective colors (white and orange) compared to the green colored capsule where the degradation product ion was detected in very low levels. Similar experiments would take hours with HPLC
UV analysis. The same study also demonstrated that a similar improvement in throughput could be achieved for the determination of the specific activity of radiolabeled compounds used in drug metabolism studies. Four isotopically labeled active ingredients showed very similar specific activities in both LC
MS and DART. Less than a 5% error was observed among the four samples with DART analysis. Additionally, 4525

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Analytical Chemistry DART did not require the addition of a solvent modifier for efficient ionization like in ESI. As a result, there was almost no radioactive waste to be concerned with after DART experiments. Metabolite screening has been targeted by DART for rapid investigation of volatile and nonvolatile compounds. An insect hormonal regulator in insects, juvenile hormone III and its corresponding terpene precursors farnesol, farnesoic acid, and methyl farnesoate, were detected to create a quick screening method for insect terpenoids.144 Careful optimization of DART conditions resulted in detection limits ranging from femtomole to subpicomole levels with accurate mass determination below 5 ppm. These merits would allow for detection of the compounds from the endogenous production in most insects. In-source CID was used to distinguish between two farnesol isomers, an important merit since only one isomer is biologically active. When orifice 1 voltages were set between 40 and 80 V, the breakdown curves revealed that the fragmentation efficiency of the EE-farnesol isomer was greater than the ZZ-farnesol isomer. The differences in fragmentation highlight the different configurations of the carbon
carbon double bond at C-2 of farnesol. Traditional metabolomics screening methods using LC
MS and GC/MS require significant time to conduct the analysis. Alternative rapid techniques may allow for larger sample sets and replicates to be screened if the sample prep steps can be parallelized. In this context, DART human serum metabolomic fingerprinting was first developed and optimized145 before implementation in a broader ovarian cancer detection study.146 Optimized DART runs required as little as 1.2 min; however, it should be noted that the method still required derivatization to increase the volatility of sample constituents. Throughout DART runs, more than 1500 different spectral features could be detected. These spectral features do not individually represent separate metabolites as some are most likely fragment and various adduct ion species. However, the quantity and the resulting detected overall peak patterns can all be used in building metabolite screening databases. Most importantly, a 4.1
4.5% repeatability was obtaining using a custom built sampling arm to introduce samples for DART ionization. In a pilot study, DART metabolomic fingerprinting of blood serum samples from 44 patients diagnosed with serous papillary ovarian cancer (stages I
IV) and 50 healthy patients with or without benign conditions were differentiated.146 Samples were subject to protein precipitation and derivatized by silanization prior to analysis. The predictive performance of the DART mass spectra was evaluated through a 64
30 split validation with and without different feature selection methods and with leave-oneout cross validation. Modified support vector machine models were used to obtain predictive rules. A total of 153 estimated elemental formulas were obtained by accurate mass measurements which were tentatively mapped to 25 metabolic pathways. The method was able to distinguish between cancer and control groups with 99
100% accuracy. This study demonstrated the potential powerful role that DART screening can have as a diagnostic tool, but more samples are required to further validate these findings and to translate this approach into a routine clinical tool. FAPA. FAPA was first described in the peer-reviewed literature under the name flowing afterglow-atmospheric pressure glow discharge (APGD),147,148 and was initially introduced at Pittcon 2005, months after DART was first introduced. Although DART and FAPA share many similarities, these two techniques have some important differences: (a) in FAPA plasma species are not

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filtered by any electrodes before interaction with the sample, leading to a more rich plasma chemistry and potential for additional reactions, (b) FAPA is operated in the glow-to-arc i
V regime (∼25 mA) where DART is operated in the coronato-glow regime (2
5 mA), and (3) heating of the FAPA gas stream is not achieved with a resistive heating element as in DART but through Joule heating within the electrical discharge. Efforts by the Hieftje group to compare these two techniques on the basis of reagent ion production, response to a model analyte, infrared thermography, and spatial emission characteristics revealed even more profound differences.149 It was found that when the FAPA source was operated in a “DART-like” mode, mainly protonated water clusters (H2O)nHþ with n = 2
7 were observed in the background spectra, whereas in the FAPA mode N2þ, NOþ, O2þ, and (H2O)nHþ with n = 2
3 were the main species. Interestingly, the abundance of protonated water clusters was lower in DART-like mode, which could have implications in terms of ion suppression effects on very complex mixtures or mixtures with a wide range of concentrations. The presence of N2þ, NOþ, O2þ 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. Charge-transfer reagent ions have also been reported in the DART background mass spectra, showing an increase in the relative abundance in O2•þ when the DART ion source was positioned closer to the mass spectrometer sampling orifice and the exit grid potential was increased from 250 to 650 V.114 Comparative thermography studies carried out with a FAPA source operated in DART-like or FAPA-like modes showed that the discharge gas was much hotter (∼235 vs 55 °C) in FAPA mode. However, these results are not directly transferable to DART experiments, as in DART an auxiliary heater is used which was not used in these studies. Time-resolved studies on the ionization of ferrocene showed that in DART-like mode, the progressive cooling of the ion source following the change in the discharge current from glow-to-arc to corona-to glow regimes resulted in a progressive increase in the number of higher-order clusters, which are less acidic, and a decrease in ionization efficiency. Most likely this effect would only be observed in the absence of an external heater, as with a FAPA source operated in DART-like mode, but not for a DART source which normally runs at much higher effective temperatures (150
250 °C) at the ion source outlet.111 In a recent study, ion suppression effects and absolute sensitivities in FAPA have been compared to those in LTP and DART using an identical mass spectrometer as detector.150 Because no chromatographic or electrophoretic steps are used in ambient sampling/ionization experiments, ionization suppression could potentially be more significant than in hyphenated techniques where the sample complexity is reduced at the separation stage. Controlled experiments carried out at various matrix-to-analyte ratios showed that ionization suppression effects followed the order FAPA < DART < LTP, where internal energy deposition followed the LTP > FAPA > DART trend. This study also showed that absolute sensitivity for methyl salicylate was up to 5 times greater for FAPA than for the other two techniques. Several interesting applications for FAPA have been described. In recent work, the Zenobi group has demonstrated the use of FAPA coupled to PCA for the fast fingerprinting of bulk bio-, homo-, and copolymer surfaces, which had not yet been demonstrated by other ambient techniques.151 FAPA has also been 4526

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Analytical Chemistry coupled to a Mattauch-Herzog mass spectrograph,152 allowing the detection of hydride-forming elements (As, Ge, Sb) through standard generation techniques used routinely in atomic spectrometry.153 Hydride generation is usually coupled to inductively coupled plasmas, glow discharges, and flames to induce atomization prior to detection. These approaches carry the potential problem that injection of large amounts of hydride species may disrupt the plasmas, which can be successfully circumvented by the use of a FAPA cell instead. The use of FAPA as an ion source for GC/MS has also been demonstrated.154 This approach has the advantage of mitigating the aforementioned ion suppression problems but at the cost of decreasing sample throughput, which is against the philosophy of most ambient ionization/sampling methodologies. However, a compromise between adequate analysis speed and improved tolerance to matrix effects was obtained by coupling FAPA to TOF mass spectrometric detectors and making use of fast chromatographic separations (under 5 min). Another interesting development in FAPA instrumentation is the “miniFAPA” ion source used for coupling capillary electrophoresis (CE) separations to mass spectrometric detection.155 The most common commercially available CE
MS interfaces are based on ESI following a (a) sheath-flow interface, (b) sheathless interface, or a (c) liquid junction interface. The sheath-flow interface is by far the more popular design but suffers from dilution of the CE effluent and a concomitant reduction in sensitivity. Also, coupling CE to ESI requires careful balance of the current from two separate power supplies to maintain efficient separation and stable ionization. In the “miniFAPA” CE
MS interface, the CE effluent was sprayed using a sonic spray-type nebulizer,156 and the effluent mist intersected with the FAPA plasma at a ∼45° angle enabling independent control of ionization and separation voltages with relatively stable operation. LTP and DBDI. These two appealing techniques make use of an unheated plasma that interacts directly with the sample. This plasma is generated by a dielectric barrier discharge produced in millimeter-sized gaps in various possible configurations. Dielectric barrier discharges (DBDs, also known as silent discharges), first developed for industrial O3 generation in the 19th century, make use of the novel feature that the electrodes are positioned outside of the discharge chamber and not in contact with the plasma.157 These types of discharges are characterized by the generation of ‘‘cold’’ nonequilibrium plasmas at atmospheric pressure and the strong influence of the local field distortions caused by space charge effects. In DBDs, one or both discharge electrodes are separated by or coated with a dielectric material such as glass, ceramics, or polymers. Because this material cannot pass a direct current, the electric field in the gap has to be high enough to produce gas breakdown. At high frequencies (5
10 MHz), the dielectric material is less efficient at limiting the current; thus, this is the preferred mode of operation to limit power consumption. The initial description of DBDs for the ionization of organic compounds received the name DBDI158 for an ion source in a point-to-plane configuration where the sample substrate also acts as the dielectric. This approach was later followed by an annular DBD configuration that received the name LTP and was allowed for directing the plasma toward specific points in space that allowed the analysis of samples of various sizes and on various substrates.159 Several applications of these two techniques have been recently described. Zhang et al. reported an initial study on

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the detection of explosives using LTP.160 Garcia-Reyes et al. later reported the comprehensive evaluation of the analytical performance of LTP-MS for the detection of explosives and related compounds.161 PETN, TNT, RDX, Tetryl, HMX, hexamethylene triperoxide diamine (HMTD), 2,4-dinitrotoluene, 1,3-dinitrobenzene, 1,3,5-trinitrobenzene, 2-amino-4,6-dinitrotoluene, 4-amino-2,6-dinitrotoluene, 2,6-dinitrotoluene, and 4-nitrotoluene were tested in the range 1 pg to 10 ng in negative-ion mode with limits of detection in the low picogram range. Sensitivity was maximized when using glass substrates heated to 120 °C. Ions typically formed from these molecules by LTP included [M þ NO2]
, [M]
, and [M
NO2]
. A “reactive” LTP mode was also successfully demonstrated in this work by addition of trifluoroacetic acid vapors to the LTP to create specific ions by reactions with RDX and HMX. The versatility of LTP has been thoroughly demonstrated for a series of applications. Jackson et al.162 used LTP for the analysis of 14 drugs of abuse such as amphetamine, cocaine, diazepam, heroine, etc. in saliva, urine, and hair extracts, reaching detection limits as low as 1 ng mL
1. Direct analysis of olive oil without pretreatment by LTP
MS/MS allowed the detection of free fatty acids (oleic, linoleic, palmitic), bioactive phenolic compounds (tyrosol, hydroxytyrosol, etc.), and volatiles (hexanal, 2-hexen-1-ol).163 A total of 13 agricultural chemicals (ametryn, amitraz, atrazine, buprofezin, DEET, diphenylamine, ethoxyquin, imazalil, isofenphosmethyl, isoproturon, malathion, parathion-ethyl, and terbuthylazine) in fruit peels and extracts were also studied by LTP
MS.164 LTP ionization coupled to MS/MS experiments was also used to detect and quantitate melamine in milk powder down to part per billion levels,165 with later work demonstrating a similar application by coupling LTP to a miniature ion trap mass spectrometer.166 A DBD atmospheric pressure ion source in a configuration similar to the LTP probe was also used for detecting hydrogen peroxide in ambient air through the detection of the [H2O2 þ O2]
ion.167 Several interesting instrumental developments involving DBDs have been recently reported. In one case, enclosed DBD was coupled with a neutral desorption (ND) approach, similar to that used in ND-EESI.168 Helium metastables were used for detection of volatiles from lemon, garlic, onions, and cinnamon, nicotine from cigarettes, pharmaceutical active ingredients from tablets, and nanogram to picogram levels of the explosive compound HMTD. Another report described a graphite DBDbased LTP ion source with two switchable operation modes with significant differences in in-source fragmentation characteristics useful for the identification of organic compounds:169 (a) a point to plane mode and (b) annular mode. Intense [M þ 2]þ and [M þ 16]þ ions for ethylbenzene and toluene and [M þ 16]þ oxidation products were observed in both modes but with the second mode inducing more fragmentation. This type of ionic species had been previously observed when a helium-supported LTP probe was used for ionization of benzene and other aromatic compounds following a mechanism involving a surface-assisted reduction pathway.170 Another interesting feature of this dual mode source is that it uses disposable graphite electrodes that can also serve the dual role of preconcentration substrates for volatile compounds. Although it may seem as if LTP/DBDI approaches would not be well suited for imaging applications due to the relatively larger size of the projected plasma plumes, it has been shown that spatially resolved measurements are indeed possible with modest lateral resolution. Zhang et al. reported on the use of LTP 4527

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Analytical Chemistry coupled to inductively coupled plasma MS for depth profiling of thin layer coatings of electronics.171 SEM images revealed an LTP-ablated spot indicating lateral resolutions of ∼200 μm. LTP
MS was also recently used for the imaging of Chinese paintings and calligraphy using a modified probe version with a smaller capillary (150 μm i.d.) that could be cooled with liquid nitrogen to minimize surface damage.172

’ MICROPLASMAS One interesting development in the field of plasma ambient ionization methods is the use of microfabricated microplasmas.173 In the work by Symonds et al., ambient ionization using microplasmas was shown to be a promising approach with some advantages over traditional plasma ambient ionization techniques.174 The microplasma used in this case was configured in a microhollow cathode (MHC) type architecture. The device was fabricated from a 525 μm thick, 100 mm diameter p-type silicon (100) wafer. An 8.2 μm SiO2 layer was deposited onto the polished side of the Si wafer, followed by a 200 nm thick aluminum electrode on top of the SiO2 layer. A 200 μm circular opening was made through the layered structure using standard plasma etching techniques. The helium discharge gas was delivered at a flow rate