Department of Chemistry, National Sun Yat-Sen University, Kaohsiung, TaiwanCancer Center, Kaohsiung Medical University, Kaohsiung, Taiwan
r t i c l e i n f o
rticle history:eceived 1 March 2011eceived in revised form 7 June 2011ccepted 7 June 2011vailable online 22 June 2011
eywords:
a b s t r a c t
Ambient ionization is a set of mass spectrometric ionization techniques performed under ambient con-ditions that allows the direct analysis of sample surfaces with little or no sample pretreatment. Usingcombinations of different types of sample introduction systems and ionization methods, several noveltechniques have been developed over the last few years with many applications (e.g., food safety screen-ing; detection of pharmaceuticals and drug abuse; monitoring of environmental pollutants; detection ofexplosives for antiterrorism and forensics; characterization of biological compounds for proteomics and
mbientesorption/ionizationwo-step ionizationlectrospray ionizationtmospheric pressure chemical ionization
metabolomics; molecular imaging analysis; and monitoring chemical and biochemical reactions). Elec-trospray ionization and atmospheric pressure chemical ionization are the two main ionization principlesmost commonly used in ambient ionization mass spectrometry. This tutorial paper provides a reviewof the publications related to ambient ionization techniques. We describe and compare the underly-ing principles of operation, ionization processes, detecting mass ranges, sensitivity, and representativeapplications of these techniques.
Min-Zong Huang received his Ph.D. in the Depart-ment of Chemistry, National Sun Yat-Sen University(NSYSU), Taiwan in 2008. His dissertation aimed at thedevelopment of electrospray laser desorption ioniza-tion mass spectrometry (ELDI-MS), applying ELDI-MSfor the direct analyses of chemical or biochemicalcompounds on solid and liquid sample surfaces, andmolecular profiling and imaging of biological tissuewith ELDI-MS. He is current a postdoctoral researcherin the Department of Chemistry, NSYSU and his recentresearches focus on the development of new samplingand ionization techniques for ambient mass spec-trometry and applying them for real-time monitoringchemical reactions.
∗ Corresponding author at: Department of Chemistry, National Sun Yat-Sen Uni-ersity, Kaohsiung, Taiwan. Tel.: +886 7 5253933; fax: +886 7 5253933.
Sy-Chyi Cheng received his Ph.D. in the Department ofChemistry, National Sun Yat-Sen University in 2010.His dissertation focused on the development of laser-induced acoustic desorption/electrospray ionizationmass spectrometry (LIAD-ESI-MS) and the applica-tions of LIAD-ESI-MS to rapidly characterize chemicaland biochemical compounds in solid or liquid states.He is now an associate researcher in the Institute ofForensic Medicine, Ministry of Justice, Taipei, Taiwan.His recent research focuses on the combination ofThin Layer Chromatography with LIAD-ESI-MS andapplying it in the fields of forensic science, food safetyand environmental analysis.
Yi-Tzu Cho is now a fifth year graduate studentin the Department of Chemistry, National Sun Yat-Sen University, Kaohsiung, Taiwan. Her researchfocuses on combining multivariate statistics withambient ionization mass spectrometry (mostly onliquid-electrospray laser desorption ionization massspectrometry) for rapid diagnosis of diseases; char-acterization of biochemical compounds in various
biological fluids; real-time monitoring organic or bio-chemical reactions; and studying protein structure byH/D exchange.
Jentaie Shiea received his Ph.D. degree in Analyti-cal Chemistry at Montana State University. He is nowserving as a professor in the Department of Chem-istry, National Sun Yat-Sen University, Kaohsiung,Taiwan. He is current president of Taiwan Society forMass Spectrometry, the executive board member andthe representative of Region B (Asia and Oceania) inInternational Mass Spectrometry Foundation and thefellow of the Royal Society of Chemistry. His researchinterests include analytical chemistry with a stronginvolvement in biological and organic mass spectrom-etry, instrumentation (especially in developing novelambient ionization sources), separation, proteomics,biomedicine, and polymer science.
. Introduction
Ambient ionization mass spectrometry, which has witnessed flurry of recent developments, is a set of useful techniquesor the analysis of samples under open-air conditions. It allowsirect, rapid, real-time, and high-throughput analyses with lit-le or no sample pretreatment [1–4]. The technique allows thenalyses of a wide range of substances from various surfaces andatrices. With its wide selection of potential desorption and ioniza-
ion methods, ambient ionization mass spectrometry allows bothighly polar/nonvolatile and nonpolar/volatile analytes to be ion-
zed and detected. In most ambient ionization mass spectrometryystems, the atmospheric ionization techniques-electrospray ion-zation (ESI) and atmospheric pressure chemical ionization (APCI)re the predominant ionization processes; analytes are mostly des-rbed through laser desorption, thermal desorption, or impact byons or charged droplets [5–7]. Since the analytes are ionized by the
echanisms of ESI or APCI, the mass spectra of ambient ionizationass spectrometry are nearly identical to that by conventional ESI
r APCI. Because the sampling processes of most ambient ioniza-ion techniques are essentially noninvasive, they are ideal tools fornvestigating the surface compositions of various biological solids.o date, more than 30 ambient ionization techniques have beeneveloped and described in the literature. With the developmentf new variants, combinations, and hybrids, several different termi-ologies and acronyms have been adapted (Tables 1–3). A numberf review articles have attempted to classify or categorize the exist-ng ambient ionization methods in terms of their differences onesorption/ionization processes or sampling/ionization processes1–4,8–15]. However, most of these reviews describe and summa-ize the fundamental principles, operation, instrumentation, andpplications of these techniques related to solid sample analysis.ith a different view, several electrospray-related ionization tech-
iques suitable for direct liquid and gas sample analysis withouthe need of pretreatment are also included.
In this tutorial paper, we further discuss the large field of ambi-
nt ionization mass spectrometry that has been published to dateith its different designs and combinations. We further sorted
mbient ionization techniques and those we have reported intohree main analytical strategies: (a) direct ionization, where analyte
able 1ummary of direct ionization techniques.
Technique name Acronym Sample state Analyte polarity
Direct electrospray probe DEP Liquid Polar
Probe electrospray ionization PESI Liquid Polar
Paper spray ionization PSI Liquid Polar
Droplet electrospray ionization Droplet ESI Liquid Polar
molecules in the liquid are directly ionized without pretreatment ina high electric field; (b) direct desorption/ionization, where chargedreactive species (e.g., ions, charged solvent droplets) or metastableatoms generated by an ambient ionization source are brought to thesample surface for desorption and ionization; (c) two-step ioniza-tion, where analyte molecules are desorbed using laser irradiation,shock waves, thermal energy, or nebulization and then broughtto the ambient ionization source for post-ionization (Fig. 1). Foreach technique, we describe, as best known, the underlying princi-ples of operation, the possible ionization processes, representativeapplications. In addition, the comparable tables including the sam-ple state, analyte polarity, detection limit and mass range are alsopresented.
2. Direct ionization
One of the simplest ambient ionization processes for liquid sam-ple analysis without sample pretreatment is direct ionization ofthe sample solution in a high electric field. ESI, an atmosphericionization method, can be used to directly ionize analytes in liq-uid samples; it is an effective means of analyzing both largebiomolecules and small organic compounds [5]. Because a capil-lary is required to conduct a small flow of the sample solution to thehigh electric field for conventional ESI, potential clogging of the cap-illary requires that the sample solutions be carefully cleaned andfiltered. Problems are, therefore, encountered when analyzing traceamounts of samples because it is impossible to clean or filter thesample without dilution and decreasing the detection sensitivity.Several designs have been developed to avoid this inconvenience,usually by inducing ESI directly from a trace sample solution ordroplet (Fig. 2 and Table 1). One approach is to employ an ion-ization source capable of producing an electrospray directly fromthe untreated sample solution, which is pre-applied on the sam-ple substrate without the need for passage through a capillary.Such techniques are known as direct electrospray probe (DEP) [16],probe electrospray ionization (PESI) [17], or paper spray ioniza-tion (PSI) [18]. Another approach is to induce ESI directly from asuspended micro-droplet; such techniques include droplet elec-trospray ionization (Droplet ESI) [19] and field-induced dropletionization (FIDI) [20].
The conceptual idea of direct ionization can be realized by usingDEP to induce ESI from an untreated sample solution pre-applied onthe probe without the need of a capillary [16]. The functions of theprobe are to (i) hold the sample solution and (ii) conduct the highvoltage for the electrospray. A probe can be simply a thin copper orplatinum ring, a copper coil with or without optical fibers or solidphase microextraction fibers in it, an optical fiber coated with a thingold or Nafion film, or a tungsten wire modified with a tungstenoxide nanowire on its surface [16,21–23]. As revealed in Fig. 3(a),the analytical procedures for a DEP-MS analysis are quite simple:the sample solution (ca. 50 nL−1 �L) is applied via micropipette tothe tip of the probe; electrospray is subsequently generated from
Highest mass Detection limit/Sample Dynamic range Reference paper
66 kDa 5 fmol/angiotensin I n.a. [21,23]12 kDa 40 amol/cytochrome c n.a. [17,25]12 kDa 270 fmol/heroin 104 [18,29]300 Da n.a. n.a. [19]2 kDa n.a. n.a. [34]3.5 kDa 10 pmol/angiotensin I 102 [36]
the solution while a high voltage (typically 2.0–4.5 kV) is applied tothe probe; the analyte ions are detected by the mass spectrometerattached to the DEP. The use of DEP-MS for untreated liquid sam-ple analysis has several unique analytical features: (i) consumption
Fig. 1. Logical and methodological categorization of the schemes involved in ambient ionization mass spectrometry. (a) Direct ionization (directly ionizing analyte moleculesi etasts les arn ).
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n an electric field), (b) direct desorption/ionization (charged reactive species or murface for desorption and ionization), and (c) two-step ionization (analyte molecuebulization and then brought to the ambient ionization source for post-ionization
f the sample solution is low; (ii) sample switching is immedi-te; (iii) a capillary and syringe pump are unnecessary (clogging ofapillary—possible in conventional ESI—will not occur); (iv) manu-acture of the probe is simple and economical. In addition, the probes extremely easy to clean and does not require maintenance. Thisechnique has been applied to the characterization of peptides androteins in several aqueous solutions [16,21–23].
Probe electrospray ionization, another direct ionization method,enerates an electrospray from a sample solution attached to ane needle probe. This technique has been applied to the analy-es of several different liquid samples [17]. In PESI, a step motorscillates a thin stainless-steel needle (c.a. 4 �m in diameter.) at
frequency of 3–5 Hz along the vertical axis. Small amounts ofhe sample solution (as low as the sub-picoliter level) are repeat-dly loaded onto the probe as it touches and leaves the sample
olution. When the probe presenting the sample solution moveso the pinnacle (e.g., to the entrance of the MS inlet of a masspectrometer), a high voltage is applied to the probe and an elec-rospray is induced from the sample solution. Compared with
ig. 2. Simplified view of the categorization of direct ionization techniques. First row (gblue): technique names and acronyms. (For interpretation of the references to color in th
able atoms generated by an ambient ionization source are brought to the samplee first formed through laser irradiation, shockwave treatment, thermal energy, or
conventional ESI, PESI has the advantages of high salt tolerance,automatic and rapid sampling capability, and extremely low sam-ple consumption. PESI-MS has been applied to (i) characterizeamino acids, peptides, proteins, and polyethylene glycol (PEG) indifferent aqueous solutions; (ii) detect biological compounds inurine, mouse brain, mouse liver, salmon egg, and fruit juices; and(iii) perform molecular imaging and real-time reaction monitoringanalyses [17,24–26].
Paper spray ionization is another recently developed direct ion-ization method [18]. The electrospray is induced from the sharptip of a triangular filter paper wetted with a small amount of solu-tion. The high voltage required for the electrospray is introducedto the solution absorbed in the paper through solution conduction.Notably, a similar design had been reported previously for an over-run TLC-MS technique where the end of the TLC plate was cut to
a sharp end and the electrospray was induced from the tip of theTLC plate after applying a high voltage to the solution on the TLCplate (Fig. 3(b)) [27]. A stable electrospray has also been inducedfrom the solution flowing out of a small open channel in a plas-
reen): ionization strategy; second row (orange): electrospray method; third rowis figure legend, the reader is referred to the web version of the article.)
Fig. 3. Schematic representations of the direct ionization of aqueous s
ic plate with a sharp triangular end [28]. PSI is useful for the directnd rapid analyses of a wide variety of compounds—including smallrganic compounds, peptides, proteins, and drugs in whole bloodr urine—absorbed in paper without sample pretreatment [18,29].
Another way to perform direct ionization of the sample solutions to generate the electrospray from the sample droplets located in
high electric field [30]. This situation can be achieved by (i) mov-ng the sample droplet close to the electrode upon which the higholtage is applied or (ii) suspending the sample droplet within theigh electric field created by two electrodes. Droplet ESI mass spec-rometry is a technique for producing analyte ions when the sampleroplet is moved through a high electric field [19,31]. A piezoelec-ric vibrator is used to generate a stream of neutral sample dropletsdiameter: ca. 50 �m; Fig. 3(c)). The droplet surface is charged as itasses through an electrode applied with a high voltage (e.g., 3 kV).he droplet is subsequently destabilized by the charges inducedn the surface and emits a fine spray of offspring droplets. Thenalyte ions produced through such droplet fission are detectedy a mass spectrometer. The polarity of the resulting analyte ionsepends on the polarity of the high voltage applied to the elec-rode. For example, positively charged analyte ions are producedhen a positive high voltage is applied to the electrode and theS inlet is grounded. Although the Droplet ESI mass spectrometry
rovides ESI-like mass spectra, its sensitivity is lower than that ofonventional ESI-MS because the electrospray can be induced onlyrom those droplets that move very close to the small electrodeurface. In addition, the number of the sample droplets capable of
enerating an electrospray is much lower than that in conventionalSI.
FIDI was developed to produce both positive and negative elec-rosprays simultaneously from a single droplet suspended between
ns using (a) DEP, (b) Overrun TLC-ESI/MS, (c) Droplet ESI, and (d) FIDI.
two electrodes (Fig. 3(d)) [20,32]. Under the influence of thehigh electric field created by the two electrodes, the suspendeddroplet becomes elongated parallel to the fields on both sides; twoelectrospray jets are emitted from the opposing conical tips to pro-duce positive and negative ions simultaneously. Again, much likeDroplet ESI-MS, FIDI-MS is not practical for real sample analysisbecause of its low sensitivity. This problem can be overcome tosome degree by employing a modified FIDI technique. The sam-ple solution flowing out of a stainless-steel capillary accumulatesas a droplet that is positioned between two electrodes bearinga high applied voltage (+6 kV at one electrode, 0 V at the other).The solution in the capillary itself bears an applied voltage of+3 kV. Electrosprays are induced from both sides of the dropletmaintained in a stationary position at the outlet of the capillary,thereby producing fine droplets of oppositely charged ions. ThisFIDI-MS has been applied to the real-time monitoring of chemi-cal and biochemical reactions [33,34]. Nevertheless, simultaneousdetection of ions of opposing polarities requires attachment of theFIDI source to two mass analyzers operated in different polaritymodes. Recently, this concept was applied to a vacuum-operatedmatrix-assisted laser desorption/ionization mass spectrometry(MALDI-MS) system equipped with two time-of-flight (TOF)mass analyzers positioned on opposite sides of the ion source[35].
Ultrasound ionization (USI) was recently reported; this newdirect ionization method uses a simple piezoelectric device to gen-erate ultrasound and thereby produce charged micro-droplets from
sample solutions for the ionization of biomolecules [36]. Quiteunlike Droplet ESI processes, the main ionization mechanism ofUAESI involves ultrasound-induced cavitation followed by ESI fromthe sample droplets. This approach allows the generation of analyte
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Table 2Summary of direct desorption ionization techniques.
Technique name Acronym Sample state Analyte polarity Highest mass Detection limit/Sample Dynamic range Reference paper
ons from proteins, saccharides, and fatty acids in aqueous solutions36].
. Direct desorption and ionization
Direct desorption/ionization (DI) of chemical and biologicalompounds by impinging sample surfaces with ions, atoms, orlusters has been an active research area ever since the develop-ent of secondary ion mass spectrometry (SIMS) and fast-atom
ombardment (FAB) in the early 1980s [37,38]. The developmentsn direct DI techniques in recent years have been quite simi-ar to those from earlier times, except that the samples are nowypically analyzed under native conditions, without manipula-ion or pretreatment; in addition, most importantly, the analysiss performed under ambient conditions. The novelty of a directI concept is the use of different charged species or photons to
mpact the sample surface under ambient conditions. The imping-ng subjects for direct DI can be (i) charged solvent dropletsenerated by ESI, (ii) bipolar solvent droplets generated by SSI,iii) singly charged solvent ions or metastable atoms generatedy APCI, and (iv) photons from laser irradiation. Analyte ions areormed rapidly after the impinging subjects impact the sampleurface.
Just like other ambient ionization processes, direct DI tech-iques have the advantage of allowing rapid and sensitive analysesf samples in their liquid or solid state. Many direct DI tech-iques have been developed in recent years. To provide a betternderstanding of these techniques, we have divided them intohree sub-categories based on the nature of the impinging sub-ects (Fig. 4 and Table 2). The ions of large analytes (e.g., peptides,roteins) are readily formed when the sample surface is impactedy charged or neutral droplets; in contrast, only small analyte
ons will be formed when the sample surface is impacted byingly charged solvent ions, metastable atoms, or photons. Thenalytes must possess a certain degree of polarity if they areo be ionized through direct DI methods—with the exceptionf those techniques involving metastable atoms, where analyteons of low polarity (e.g., aromatic compounds) can also beroduced.
.1. Direct DI by impinging sample surface with droplets: DESI,eactive-DESI, GI-DESI, TM-DESI, DeSSI, EASI, and EADESI
Desorption electrospray ionization (DESI), uses charged sol-ent droplets for the sampling and ionizing of analytes on sampleurfaces (Fig. 5(a)) [39]. For DESI, the sample surface (usuallyntreated) is impinged, by charged solvent droplets generated from
pneumatically assisted electrospray emitter, at an angle relativeo the sample surface and mass spectrometer inlet; the analyteons are produced as a result of interactions of the charged solventroplets with the desorbed analyte molecules. The charged solventroplets usually have diameters of less than 10 �m and impact theample surface at velocities typically exceeding 100 m s−1.
The main contributing ionization mechanism in DESI is believedo be multi-stage momentum droplet pick-up, where the sampleurface is pre-wetted by the initial impacted droplets and then theissolved analytes within the surface solvent-layer are impactedy later-arriving charged droplets to form more microdroplets.he analytes in the newly formed charged microdroplets are thenonized through electrospray processes to form singly or multiplyharged ions, which are subsequently detected by a mass spec-rometer attached to the DESI source [40–42]. ESI-like mass spectra
f polar compounds are obtained in DESI-MS (i.e., singly and/orultiply charged ions).DESI is, by far, one of the most utilized ambient ionization tech-
iques, particularly for surface analysis. Both small organic and
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large biological compounds can be characterized with detectionsensitivity comparable with that of conventional ESI. Therefore, anumber of applications of DESI for the analyses of real samples havebeen developed: the rapid analyses of pharmaceuticals, explosives,chemical warfare agents, drugs of abuse, and industrial polymers;environmental monitoring; analyses of plants and natural prod-ucts; characterization of metabolites, peptides, and proteins forproteomic and metabolomic studies; profiling and imaging of bio-logical tissues; medicinal analyses of dried blood spots; and thedirect characterization of compounds on TLC plates [39,43–51].Further, different lipids detected in the negative-ion mode by DESI-MS are successfully used to build 3D models of a mouse brain tissue[52].
Modification of the DESI technique has resulted in the devel-opment of a number of different direct DI methods, where subtlemodification leads to shifts in analytical performance. For exam-ple, reactive DESI, where charged droplets containing reactants aregenerated by electrospraying solutions doped with specific chemi-cal reagents [53]. When the charged droplets impact the samplesurface, the reactants undergo selective reactions with the ana-lytes. Hydroxylamine has been used as the reactant in reactive DESIto react with carbonyl group of steroid to enhance its detectionselectivity and sensitivity [54]. Several applications of reactive DESIfor the rapid screening of certain chemical compounds in biologi-cal fluids have been demonstrated, including the screening aminoacids, triacetone triperoxide, artesunate antimalarials, and anabolicsteroids in urine [53–56]. In addition, recognition of cis-diol func-tional groups in molecules has also been achieved through reactiveDESI [53].
Geometry-independent DESI (GI-DESI) is another type of DESIthat has been developed to improve the detection sensitivity.In this technique, the electrospray plume, sample surface, andMS-inlet capillary are all positioned within a pressure-tight enclo-sure [57]; the resulting operation and sampling with GI-DESIappears to be simpler and more efficient than that of conven-tional DESI. Transmission mode desorption electrospray ionization(TM-DESI), which employs DESI in a transmission mode, involvespassing the electrospray plume through a sample pre-depositedon a mesh substrate [58]. The approach has been used to char-acterize certain chemical compounds (e.g., thiols) applied to thesubstrate and for high-throughput screening of targeted analytes[59–61].
Another technique, referred to as electrode-assisted desorptionelectrospray ionization (EADESI), has been developed for the rapid,noncontact surface analysis of materials under ambient conditions[62]. The analyte ions are generated from the sample surfacesthrough the impact of highly charged droplets produced from thesolvent on a sharp-edged tip subjected to a high applied voltage.EADESI has been applied to the sample analyses of amino acids,peptides, proteins, and drugs in human fluids (e.g., urine, blood)[62].
Easy ambient sonic spray ionization (EASI, previously termed asdesorption sonic spray ionization, DeSSI) impinges the sample sur-face with bipolar solvent droplets generated from a (super)sonicspray promotes the efficient desorption and ionization of analytesfrom sample surfaces [63,64]. Compared with DESI, the device ofEASI is advantageous since it uses neither heating nor high volt-ages at the spray capillary [63]. However, EASI operates under aunique mechanism based on imbalanced distribution of charges onvery minute solvent droplets created from sonic spray ionization(SSI) [65]. The EASI technique has been applied to the fingerprintingclassification of perfumes, propolis, and inks; it has been coupled
with TLC for the direct characterization of separated chemical com-pounds on TLC plates [63–67]. Recently, a dual mode self-pumpingtechnique so called Venturi-EASI (V-EASI) was derived for bothsolid and liquid sample analysis [68].
Fig. 4. Simplified view of the categorization of direct desorption/ionization techniques. First row (green): ionization strategy; second row (orange): sampling/ionizationmethods; third row (blue): technique names and acronyms. (For interpretation of the references to color in this figure legend, the reader is referred to the web version ofthe article.)
Fig. 5. Schematic representations of direct desorption/ionization techniques using (a) DESI, (b) DAPCI, (c) DBDI, and (d) LSI.
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.2. Direct DI by impinging sample surface with speciesenerated by APCI: DAPCI, DBDI, LTP, PADI, APGDI and DCBI
Desorption atmospheric pressure chemical ionization (DAPCI)nvolving corona discharge ions is a direct ambient ionizationechnique for surface analysis (Fig. 5(b)) [69]. The volatile or semi-olatile compounds emitted from the sample surface upon impactith a heated gas stream are carried by the gas to react with
eagent species (e.g., electrons, protons, metastable atoms, hydro-ium ions, protonated solvent ions, etc.) generated by atmosphericressure corona discharging. The ionization mechanisms of DAPCIre similar to those in conventional APCI processes; ion/moleculeeactions (IMRs) play an essential role in the formation of analyteons. Relative to ESI, DAPCI is much more sensitive and efficientor the detection of analytes of low polarity. The DAPCI techniquellows the implementation of characteristic IMRs; it also main-ains the advantages of ambient sampling for rapid analyses ofhemical compounds on the surfaces of foodstuffs, skin, clothes,harmaceuticals, peroxide explosives, powders, and sticky liquids69–75].
Dielectric barrier discharge ionization (DBDI) is a techniquehat uses reactive plasma species for the direct DI analysis of thenalyte on solid surfaces (Fig. 5(c)). The source comprises a cop-er sheet electrode, a discharge electrode, a piece of glass slide
n between them as a dielectric barrier, and a sample plate [76].nlike those techniques using the ions generated by corona dis-harge for DI, DBDI applies an alternating voltage of 3.5–4.5 kVt a frequency of 20.3 kHz to two electrodes separated by a glasslide; when an inert gas (e.g., He) flows between the electrodes,
stable low-temperature plasma is formed. The analytes on theurface of the glass slide are desorbed and ionized by impinginghe sample surface with the reactive plasma species. The singlyharged analyte ions are formed mainly through Penning ioniza-ion; metastable He atoms generated in the plasma react withhe analytes to form singly charged analyte ions [76]. DBDI cou-led with MS has been applied for the characterization of smallrganic compounds in several real samples. For example, detect-ng trace amounts of explosives on various surfaces (paper, cloth,hemical fiber, glass, paints, soil); rapid detection of illegal drugs iniofluids; and high-throughput pharmacokinetics and therapeuticnalyses under ambient conditions without any sample pretreat-ent [76–78].The low-temperature plasma (LTP) probe used in another
lasma-based ambient DI technique comprises a glass tube, annternally grounded metal electrode centered axially, and an elec-rode surrounding the outside of the tube [79]. A discharging higholtage (2.5–5 kV at a frequency of 2–5 kHz) is used to generatehe dielectric barrier discharge. As in the cases of DBDI and PADIplasma-assisted desorption ionization), an inert gas (He, Ar, N2) orir is fed through the glass tube to facilitate the discharge processnd to transport the analyte ions to the inlet of the mass spec-rometer. The sampling plasma torch operated at low temperature30 ◦C) interacts directly with the sample being analyzed, desorb-ng and ionizing the surface molecules in the ambient environment.eal-time monitoring of chemical reactions, depth profiling ofanometer coatings, analysis of drugs of abuse in biofluids (urine,aliva), detection of explosives on solid surfaces, quality control anduthentication of virgin olive oil, food safety, homeland security,orensics, and metabolomics have all benefitted simply by direct-ng the plasma to the surface of interest, suggesting that LTP-MSan be a useful analytical tool [79–86].
Plasma-assisted desorption ionization, another plasma-based
mbient DI technique, employs a cold and non-thermal rf-plasmaeedle placed coaxially within a ceramic tube [87]. Typically, aeak-to-peak rf voltage of 200–500 V with a radio frequency of3.6 MHz is applied to the unsharpened end of a needle within a
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He gas flow. In PADI, the plasma (typically ca. 1 mm in diameterand extending up to 10 mm from the tip) is brought into directcontact with the objects for direct surface analysis. Experimen-tal results suggest that PADI/MS could be a valuable and versatiletool for high-throughput screening of small molecules in forensic,pharmaceutical, and biological applications [87].
In atmospheric glow discharge ionization (APGDI), which isbased on the establishment of a DC glow discharge in ambient airto produce plasma from the inert gas, the plasma species are drawninto a region of reduced pressure to react with the volatile organiccompounds [88]. Volatile analytes are introduced, at reducedpressure, through an orifice into the glow discharge region forpost-ionization. The flowing afterglow/atmospheric pressure glowdischarge ionization (FA–APGDI) technique uses a glow dischargein the flowing afterglow mode to produce reacting ions and excitedmetastable species from He. The DC discharge is operated in He atatmospheric pressure; the resulting species react with the analyteto ensure ionization. The analyte can be a polar or nonpolar chem-ical or biochemical compound. The technique has been appliedto characterize volatile pharmaceutical compounds and to rapidlyscreen pesticides in foodstuffs [89–92]. When employing a lasersystem for sampling, the resulting technique, laser ablation/flowingatmospheric pressure afterglow (LA–FAPA) mass spectrometry, canbe used for molecular imaging [93].
Another direct DI technique that uses the reactive species gen-erated by corona discharge is desorption corona beam ionization(DCBI) [94]. Similar to DAPCI, a high DC voltage (1–5 kV) is appliedto a sharp needle to induce corona discharge from He atoms nearthe needle. A visible corona discharging beam is observed when ahigh DC voltage is applied to the source at a low current (mA). Thereactive species (ions or metastable He atoms) generated by coronadischarge react with the analytes on the object surface to formsingly charged analyte ions. Several types of small polar/nonpolarmolecules (e.g., explosives, pesticides, estrogens, veterinary addi-tives, drugs, furfural, food additives) have been detected on solidsample surfaces using DCBI-MS [94,95].
3.3. Direct DI by irradiating sample surface with photons: LSI
The term “laser spray ionization” (LSI) has been used by dif-ferent research groups to name their techniques for generatinganalyte ions through laser irradiation of sample solutions [96,97].An LSI source has been used in conjunction with liquid chro-matography/mass spectrometry (LC/MS) to generate analyte ionsby irradiating the solution flowing through the stainless-steelcapillary with a 10.6 �m pulse infrared (IR) laser. Unfortunately,the intensity of the analyte ion signals obtained in this laser-induced thermally heating action was quite low. A much strongeranalyte ion signal was obtained, however, when a high electricfield (>500 V) was applied to the stainless-steel capillary. Indeed,for the same sample solution, the analyte ion intensity obtainedthrough this version of LSI was an order of magnitude strongerthan that obtained through conventional ESI or ion spray ionization[96,98,99].
Multiply charged ESI-like analyte ion signals have been obtainedwhen the reverse side of a glass slide pre-deposited with a dryanalyte/matrix sample spot was irradiated with a pulsed laserbeam under ambient conditions (Fig. 5(d)). The ionization mech-anism of this LSI technique appears to occur through formation ofmatrix/analyte clusters under the action of laser irradiation, evapo-ration of the matrix from the clusters, and then formation of highly
charged analyte ions through either charge condensation or ionejection processes, as in conventional ESI. The use of this LSI processto analyze liquid samples has generated both singly and multiplycharged analyte ions from small and large molecules [97,100,101].
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. Two-step ionization
Two-step ionization is performed in systems comprising a unitor producing and transferring the analyte, an ambient ionizationource, and a region in which the analytes react with charged oretastable species generated from the ambient ionization source.
he overall processes for the formation of analyte ions are dividednto two separate steps: (i) generating analyte species (i.e., ana-yte, analyte-containing particles or droplets) from liquid or solidample through the effects of nebulization, laser desorption, laserblation, thermal evaporation, thermal desorption, or shockwaves;ii) reacting the analyte species with charged solvent species or
etastable atoms generated through ESI, APCI, or photoionizationo form analyte ions. Such two-step ionization processes permithe rapid and sensitive analyses of trace chemical compounds inomplex matrices.
More than 20 different two-step ionization techniques haveeen developed (Table 3). Each technique differs from the others inerms of its sampling (including analyte production and transfer)r ambient ionization method. The analyte species can be pro-uced from liquid or solid samples subsequently they enter thewo-step ionization source to react with the reacting species, withr without the assistance of a gas stream. In this review, we classifyhe reported two-step ionization techniques into three categoriesepending on the type of sampling system. We then further classifyhe techniques in each category in terms of the ambient ionizationource used to generate the reactive species for post-ionization ofhe analyte molecules (Fig. 6).
.1. Sampling and transferring analyte through a gas stream
A gas stream is used to carry the analyte species to react withhe charged solvent species to form analyte ions. For the sensitiveetection of mixtures containing volatile and thermally stable ana-
ytes, the analytes are transferred to the ionization source using heated gas stream. Nonvolatile or thermally labile compoundsn solution can be directly nebulized to form fine droplets; theroplets are then carried by a gas stream to react with the chargedolvent species in a two-step ionization source. The chemical com-ounds in the sample solution can also be separated using HPLCnd nebulized, with the fine droplets containing the analytes thenarried by a gas stream to the ion source.
.1.1. Using charged solvent species for post-ionization ofnalyte: MC-ESI, GC-ESI, and SESI
Variations of ESI spray-based techniques have afforded a num-er of two-step ionization methods, where small experimentalhanges can lead to great shifts in analytical performance. Forxample, so-called multiple-channel electrospray ionization (MC-SI), a typical ESI system containing more than one electrosprayer,as been used to characterize analytes generated through heat-
ng. The advantage of using MC-ESI for two-step ionization is thathe ionization efficiency is greatly increased by the greater num-er of charged solvent species available for reactions. MC-ESI haseen used to real-time monitoring of organic reactions involvingolatile and thermally unstable compounds (e.g., highly reactiveetenes) by continuously carrying the volatile organic compoundsn a reaction vessel with heated N2 stream to the central channelf a seven-channel ESI source [102]. An electrospray was continu-usly generated from the surrounding six electrosprayers using ancidic methanol solution. The reactions responsible for the forma-ion of analyte ions in this MC-ESI system include (i) ion–molecule
eactions between analyte molecules and protons, hydronium ions,r protonated solvent species and (ii) the fusing of the analyte inhe charged solvent droplets with subsequent ESI from the analyte-ontaining droplets [102].
ica Acta 702 (2011) 1– 15 9
Another application of MC-ESI is the coupling of GC with MC-ESI [103]. In this case, GC-ESI system features a GC connected toa seven-channel ESI source to separate and detect a mixture ofvolatile organic compounds. The analytes that eluted from thepacked GC column were carried by a heated N2 stream to thecentral channel of the MC-ESI source. Presumably, the chargedspecies produced by the outlying six electrosprayers reacted withthe molecules that eluted from the GC column to form the analyteions. Another GC-ESI system, featuring coupling of a capillary GCcolumn with an ESI source equipped with single electrosprayer, hasbeen used for the reproducible and sensitive analyses for volatileorganic compounds [104].
The concept of using MC-ESI for post-ionization has also beenapplied to so-called secondary electrospray ionization (SESI), whichcombines GC with an ESI source equipped with single electro-sprayer and ion mobility spectrometry (IMS) [104]. The analytesthat eluted from a capillary GC column were carried by heatedN2 to a reaction chamber where they reacted with charged sol-vent species generated by ESI; the resulting ionic species werethen directed to an IMS system for further desolvation and ioniza-tion. This technique has been applied to characterize the chemicalcompounds in illicit drugs, explosive vapors, and chemical war-fare agent stimulants [105–107]. Recently, it has also been usedto detect volatile organic compounds from bacterial cultures[108].
4.1.2. Using charged solvent droplets for post-ionization ofanalytes in droplets: FD-ESI, EESI, and ND-EESI
The two-step ionization technique known as fused droplet elec-trospray ionization (FD-ESI) was developed to generate peptide andprotein ions from aqueous solutions (Fig. 7(a)) [109]. In FD-ESI,droplets containing analyte species are generated from the samplesolution by an ultrasonic, pneumatic, or piezoelectric nebulizer. Thedroplets are delivered in a N2 gas stream to an ESI plume for post-ionization [109–113]. The ionization process responsible for theformation of analyte ions involves the analyte-containing dropletsfusing with the charged solvent droplets generated by ESI and thenESI subsequently proceeding from the newly formed droplets toform analyte ions. Because the composition of the ESI solution canbe changed, analytes with different polarities could, in theory, bedetected selectively by varying the polarity of the ESI solution. Ithas been found that FD-ESI has much higher salt tolerance thandoes conventional ESI. For example, when using methanol as theESI solvent, interference from small organic and inorganic com-pounds (e.g., NaCl, NH4Cl, phosphates, Tris) in the sample solutionis readily removed through FD-ESI. Because the solubility of thesesalts in methanol is low, the analyte species are transferred selec-tively to the charged methanol during droplet fusion processes[111–113].
Extractive electrospray ionization (EESI) employs two separatesprayers that similar to FD-ESI: one to nebulize the sample solu-tion and the other to produce charged solvent droplets through ESI[114]. The analyte-containing droplets are carried by a N2 streamto meet with the charged solvent droplets generated by ESI. Theanalyte molecules in the sample droplets are extracted into thecharged solvent droplets and then ESI proceeds from the fuseddroplets to form analyte ions. EESI has been applied to the detec-tion of melamine in untreated milk and wheat gluten, in vivobreath analysis, on-line monitoring of organic chemical reactions,wastewater analysis, and analysis of trace organic compounds inbiological fluids (urine, milk) [114–118]. Neutral desorption EESI(ND-EESI) is a modified version of EESI [119]. In ND-EESI, N2 gas
is used to desorb the analyte from the sample surface; the des-orbed analytes are then introduced into an electrospray plume forpost-ionization. ND-EESI has been used for in vivo analysis of thechemical compounds on skin and frozen meat. It has also been
Fig. 6. Simplified view of the categorization of two-step ionization techniques. First row (green): ionization strategy; second row (orange): sampling method; third row( For intt
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sed for metabolic fingerprinting of different biological samples119–122].
MC-ESI has also been used to characterize analytes eluted fromn HPLC system. The solution flowing through the HPLC columnas pneumatically nebulized and the analyte-containing dropletsere subsequently fused with the charged solvent droplets gen-
rated by ESI for further ionization [123]. One of the advantagesf combining MC-ESI with HPLC is that the flow rate of thePLC system can be much higher than that employed in regularPLC/MS.
.1.3. Using charged species generated by APCI forost-ionization: LPI and APPeI
Introduction of an inert gas, typically helium (He) or argon (Ar),nto a high electrical field produces a number of plasma species,ncluding radicals, Ar/He ions, protons, electrons, and, mainly,
etastable Ar/He atoms. Several ambient ionization techniques usehese plasma species for post-ionization, with Penning ionization,n which ionization of the analyte occurs through energy transferrom the excited metastable inert gas atoms, as the major mech-nism. Because only singly charged analyte ions are produced byhese plasma-based ambient ionization techniques, the analytesre usually limited to those that are small and volatile.
Liquid surface Penning ionization (LPI) is an ambient ionizationass spectrometric technique that combines Penning ionization on
he surface of a liquid using excited Ar atoms (Ar*) with subsequent
harge transfer reaction [124]. LPI employs a beam of metastabler atoms at atmospheric pressure to impact the surface of the sam-le solution, prepared by mixing the sample with a matrix (e.g.,lycerol or organic solvent) and placing it on a needle tip. Ioniza-
erpretation of the references to color in this figure legend, the reader is referred to
tion occurs when the metastable atoms impinge the sample-coatedneedle, subjected to kilovolt potentials, through Penning ionizationor IMRs between the metastable Ar atoms, the solvent/matrix ions,and the analyte. The LPI method has been applied to characterizetriorganotin carboxylates [124]. The atmospheric pressure Penningionization (APPeI) source uses a circular array of corona dischargeneedles in an Ar atmosphere to ionize gas phase samples [125].Similar to an LPI source, the high-voltage discharge needle arrayin an APPeI source is shielded from the operator for safety rea-sons. This technique is suitable for the analysis of volatile organiccompounds only; it has been applied, for example, to characterizealiphatic hydrocarbons [125,126].
4.2. Sampling by laser desorption/ablation and laser-inducedshockwaves
When analytes in solid or liquid samples are desorbed/ionizedunder irradiation of the sample surface with a pulse laser, thetechniques are known as laser desorption/ionization (LDI) ormatrix-assisted laser desorption/ionization (MALDI), if the sampleis analyzed alone or premixed with an organic matrix, respectively.The analyte can also be desorbed/ionized by the action of shock-waves generated by irradiating the rear side of a sample plate(e.g., thin metal film) with a laser pulse; this technique has beencalled laser-induced acoustic desorption/ionization (LIAD). One ofthe advantageous analytical features when using a laser beam for
desorption is high spatial resolution of the sampling area. Thisfeature allows the possibility of molecular imaging analysis andensures low sample consumption. Nevertheless, most of the des-orbed species are neutral, and their ionization efficiency in LDI,
Fig. 7. Schematic representation of a two-step ionization t
ALDI, or LIAD is usually low (<1%) [127–129]. One way to over-ome this problem is to develop a technique to efficiently ionizehese desorbed neutral species. In the last few years, charged sol-ent species or metastable atoms generated by ESI and APCI haveeen used to react with the desorbed neutral species to post-ionizehe analytes under ambient conditions.
.2.1. Using charged ESI solvent species for post-ionization: ELDI,ALDESI, LAESI, IR-LADESI, LEMS, LDSPI, and LIAD-ESI
Several ambient ionization techniques combining laser desorp-ion/ablation for sampling with electrospraying for post-ionizationf the desorbed species have been developed in the last few yearsFig. 6). The analytes are desorbed by irradiating the untreated sam-le surface with a pulse laser; the desorbed analyte species (ornalyte-containing droplets) are subsequently ionized by enter-ng an ESI plume (positioned several millimeters above the samplepot) where they react with the charged solvent species. The mainonization mechanisms in these techniques appear to be electro-pray processes, as evidenced by the formation of multiply chargedpecies from biological molecules. These techniques have severalnique analytical features: (i) intact biological molecules (pro-ein) are readily desorbed through LD, even if there is no organic
atrix—quite distinct from the effect in MALDI; (ii) pulse lasers
ith a wide range of wavelengths and frequencies can be used
or desorption (i.e., nanosecond or femtosecond pulse lasers withavelengths in the UV, Vis, or near-IR region); (iii) volatile, non-
olatile, solid, and liquid samples can all be analyzed; (iv) no
ques using (a) FD-ESI, (b) ELDI, (c) LIAD-ESI, and (d) DART.
additional interface is required to attach the ambient ionizationsource to the mass analyzer [130,131].
Electrospray laser desorption ionization (ELDI), which com-bines the features of both ESI and LD, allows the direct, sensitive,and rapid analyses of chemical compounds on sample sur-faces (Fig. 7(b)) [130]. Both small organic molecules and largebiomolecules (i.e., proteins) are readily desorbed by a pulse laseroperated at a UV wavelength (266 nm) for nanosecond duration.The peptides and proteins in dry or wet biological media (blood,serum, saliva, tears) and the chemical components on the sur-faces of various solids (e.g., documents, paintings, compact discs,drug tablets, animal brain tissue) have been characterized directlythrough ELDI-MS without sample pretreatment. Chemical and bio-logical compounds dissolved in polar or nonpolar solvents (e.g.,water, methanol, methylene chloride, tetrahydrofuran, toluene,and hexane) can also be characterized directly by ELDI-MS throughthe assistance of fine carbon powders, which absorb the laserenergy, suspended in the solution [130–137]. In addition, with theassistance of an automatic moving stage (100 �m s−1) and the highspatial resolution (100 �m) of UV laser beams, ELDI-MS has beenused for molecular profiling and imaging of fungus and plant slicesand for the surface analysis of TLC plates [4,138–140]. ELDI-MS hasalso used to monitor the progress of chemical reactions in solu-tions of various polarities; for example, the epoxidation of chalcone
in ethanol, the chelation of ethylenediaminetetraacetic acid withcopper and nickel ions in aqueous solution, the chelation of 1,10-phenanthroline with iron (II) in methanol, and the tryptic digestionof cytochrome c in aqueous solution [135]. After adding reactive
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eagents to the ESI solution, reactive-ELDI has been used to performapid biochemical reactions (e.g., disulfide bond cleavage) duringhe fusion processes [141]. The detection limit of ELDI-MS for anntreated sample dissolved in an aqueous solution (ca. 10−7 M) isomparable with that of conventional ESI-MS (where the sampleust go through tedious sample pretreatment). The detection limit
f ELDI-MS for the analysis of compounds in the solid state is alsoomparable with that of MALDI-MS (where the selection of a suit-ble organic matrix is required and analysis is performed underacuum). One of the analytical limitations of ELDI is, however, thatnly polar compounds can be ionized in the ESI plume and detectedy ELDI-MS.
In addition to ELDI, several other hybrid ambient ionizationechniques combining laser desorption/ablation with ESI have beeneported. The main differences among these techniques are theresence or absence of matrix and the type of laser used (i.e., laseravelength or pulse). Similar to the MALDI technique, a matrix
endogenous or exogenous) is used to prepare the sample prioro LD in a technique known as matrix-assisted laser desorptionlectrospray ionization (MALDESI). High-resolution ambient ion-zation mass spectra have been obtained after combining MALDESI
ith Fourier transform ion cyclotron resonance mass spectrometryFT-ICR-MS) for biological sample analysis [142]. As in the MALDIechnique, the sample to be analyzed is mixed with the organic
atrix that absorbs the laser energy and assists desorption of thenalytes. Applications of MALDESI-MS in several biochemical stud-es have been reported, including “top down” proteomics analysesnd the direct characterization of intact proteins, polypeptides, car-ohydrates, and other biomolecules on tissue samples [142–145].
Laser ablation electrospray ionization (LAESI) employs a mid-R pulse laser for desorption [146]. Because of interactionsetween the ablation plume and the spray, LAESI also accom-lishes electrospray-like ionization. The use of a mid-IR pulse laser2.94 �m) for desorption means that the samples used for LAESI-MSnalysis must be water-rich so that they undergo high absorption ofaser energy. LAESI has been applied to characterize proteins, lipids,nd metabolites in biological fluids (urine, blood, and serum), tis-ue sections, and cells and for in vivo spatial molecular profilingnd imaging analysis [146–150]. Infrared laser-assisted desorptionlectrospray ionization (IR-LADESI), employing an IR laser beam2.94 �m) for sample desorption, has been used to characterizeharmaceutical and biological compounds in various biological flu-
ds [151]. IR-LADESI differs from LAESI only in terms of the energy1.0 mJ), and incident angle (45◦) of the laser beam. Laser electro-pray mass spectrometry (LEMS or fs-ELDI) uses a non-resonant,emtosecond-duration (70 fs) laser pulse for sample desorption; itas been applied to rapidly characterize explosives and severalharmaceutical compounds on various surfaces, including glass,abric, steel, and wood [152–154]. Laser desorption spray post-onization mass spectrometry (LDSPI-MS) has been used for the
ass spectrometric analyses of yogurt and related products [154].ifferent yogurt products can be rapidly sorted in terms of differ-nces in their LDSPI fingerprinting mass spectra [155].
LIAD-ESI-MS, an ambient ionization technique that couples LIADith ESI mass spectrometry, has been developed to characterize
nalytes in solid or liquid sample (Fig. 7(c)) [156]. LIAD-ESI dif-ers from ELDI (and its related techniques described above) in thatt uses a high-power laser pulse (e.g., 10 mJ) to irradiate the rearide of a thin (10–20 �m) metal foil presenting the sample. Shock-aves and heat are induced by the irradiation of the metal foilith the pulse laser beam, resulting in desorption of the analyte
rom the sample pre-applied on the other side of the foil. The des-
rbed analyte species enter an ESI plume for further ionization.gain, both small organic and large biological compounds, includ-
ng amino acids, peptides, and proteins (as large as albumin, m/z6,000) can be ionized and detected using LIAD-ESI-MS [156]; this
ica Acta 702 (2011) 1– 15
technique has also been applied to characterize small organic com-pounds separated on a TLC plate [157]. Because the sample is notdirectly irradiated by the laser beam, the technique is useful forthe analyses of light-sensitive compounds. In addition, because thelaser beam is positioned on the opposite side of the sample from theelectrosprayer, the ionization region and working space in a LIAD-ESI source are much larger than those in an ELDI source, makingthe operation of the LIAD-ESI source easier and safer.
4.2.2. Using APCI species for post-ionization: LD–APCI and LDTDThe analytes produced by laser desorption/ablation or shock-
waves can be ionized by reacting them with charged solvent speciesgenerated through APCI. Because the ionization processes occurmainly through IMRs between polar analytes and singly chargedsolvent species, only singly charged analyte ions are produced. Thisfeature means that the techniques in this category are only usefulfor analysis of small compounds. Because many organic compoundsof low polarity can also be ionized through reacting with the APCIspecies, the techniques in this category are useful for the analysesof polar, less-polar, or nonpolar compounds.
Laser desorption/atmospheric pressure chemical ionization(LD–APCI) uses an inferred CO2 pulse laser for desorption; thedesorbed analyte species are further ionized through reactionswith charged ion species generated by APCI [158]. LD–APCI-MShas exhibited reasonably good performance when characterizingsmall peptides present in a polyacrylamide gel after electrophoreticseparation. Indeed, LD–APCI-MS may be a potential technique forrapidly identifying proteins via peptide fingerprinting and proteindatabase searching [158,159].
Laser diode thermal desorption (LDTD) combines ultrafast ther-mal desorption with APCI. The sample desorption process in LDTDis similar to that in LIAD: the rear side of a stainless-steel sheetis irradiated continuously by an inferred diode laser for a shortperiod of time (5 s) to thermally desorb the analytes applied on theother side of the sheet [160]. The resulting analytes subsequentlyenter a corona discharge region to undergo APCI processes. LDTD-MS has been applied for qualitative and quantitative analyses ofsmall chemical compounds (e.g., drugs) in blood, plasma, urine,human liver microsomes, biological buffers, drinking water, sludge,sediment, dairy milk, and honey [160–163].
4.3. Sampling by pyrolysis, thermal desorption and heating
Other than the use of laser irradiation for desorption, analytesin solid samples can also be efficiently desorbed through pyroly-sis or heating. Because most biological compounds decompose atelevated temperatures, only thermally stable and volatile organiccompounds can be characterized using this technique. Charged ormetastable species generated by ESI and APCI have been reactedwith the thermally desorbed analyte species for post-ionization.Atmospheric pressure photoionization (APPI) is another ambientionization technique known for its capability to ionize low-polarityor nonpolar compounds. APPI has also been applied, when com-bined with a thermal desorption unit, for the rapid analyses of smalland less-polar compounds.
4.3.1. Using Charged ESI Solvent Species for Post-Ionization:ESA-Py, AP-TD/ESI, and TDAMS
Electrospray-assisted pyrolysis ionization/mass spectrometry(ESA-Py–MS) combines a pyrolytic apparatus for desorption withan ESI unit for post-ionization of the desorbed species [164]. Dur-ing ESA-Py analysis, the sample in solid or liquid state is pyrolyzed
at high temperature (i.e., 590, 630, or 940 ◦C) by a Curie-pointpyrolyzer. Similar to the operation in FD-ESI or ELDI systems, thedesorbed gaseous pyrolysates are delivered by a N2 stream to an ESIplume for post-ionization. ESA-Py has been applied to characterize
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race polar compounds that coexist with large amounts of non-olar hydrocarbons in crude oil, amber, humic substances, rubberamples, and synthetic mixed polymers [164,165].
Atmospheric pressure thermal desorption/electrospray ioniza-ion (AP-TD/ESI) is another type of thermal desorption-relatedmbient ionization technique that uses an electrospray solventlume as the ionization vehicle for thermally desorbed neutralolecules (e.g., volatile compounds, pyrolytic products) [166]. This
pproach has been used to characterize biological compounds inacterial spores. The experimental results indicate that the tech-ique allows spore detection even in the presence of a complexatrix (e.g., growth media) in crude lyophilized samples [166].Thermal desorption-based ambient mass spectrometry
TDAMS) uses gold nanoparticles (AuNPs) to assist the thermalesorption of small organic compounds and ESI for post-ionization167]. A layer-by-layer (LBL) self-assembled multilayer of AuNPsn a glass chip (Glass@AuNPs), with absorption capacity in theear-IR (NIR) region, was used as the energy absorber and sampleolder. A NIR diode laser was employed to desorb the analytes
rom the Glass@AuNPs chips and then the analytes were furtheronized in an ESI plume. TDAMS is suitable for the selectiveharacterization of small target organic compounds (small acids,mino acids, insecticides, ethyl esters in biodiesels) in complexamples [167].
.3.2. Using charged species generated by APCI forost-ionization: ASAP and DART
An atmospheric pressure solids analysis probe (ASAP) has beeneveloped to characterize volatile or semivolatile organic com-ounds in liquid or solid samples. ASAP uses a hot N2 streamo impact the sample surface [168]. The resulting analytes arehen transferred by the N2 to the APCI needle tip, where they areonized through corona discharge-based APCI processes. Severalompounds (steroids, polymers, biological tissue samples/fluids,rugs, airborne particles) have been detected using the technique168,169].
Direct analysis in real time (DART) involves ionization of volatilerganic compounds originated from gas, liquid, or solid samples.t is useful for the rapid and noncontact analysis of materials inpen air (Fig. 7(d)) [170]. The ionization process in a DART sources based on the reactions of electronic or vibronic excited statepecies with reagent molecules and analytes (or so-called Penningonization). Such ionization events have been performed from a
ide variety of the sample surfaces: concrete, asphalt, human skin,irline boarding passes, business cards, fruits, vegetables, spices,everages, cocktail glasses, currency bills, tablets, bodily fluidsblood, saliva, urine), plant leaves, fruits, vegetables, and clothing.ART/MS allows the ready characterization of both polar and non-olar organic compounds, including explosives, active ingredients
n pharmaceutical tablets and capsules, metabolites in urine, fattycid methyl esters in bacterial cells, and chemical warfare agents170–178]. The technique has also been used to characterize smallrganic compounds separated on TLC plates; coupled with HPLCeparation for sample analysis; and employed in real-time analysisor reaction monitoring in drug discovery [179–181].
.3.3. Using photons and charged species generated by APPI forost-ionization: DAPPI
Desorption atmospheric pressure photoionization (DAPPI)nables the direct analysis of volatile organic compounds in solidamples [182]. A confined heated solvent vapor (e.g., toluene) issed to thermally evaporate the analyte from the sample surface.
he desorbed analytes are consequently ionized through multi-hoton ionization by irradiating the analyte with UV light or by
MRs between the analyte and the charged species generatedhrough photoionization of water molecules in air. These ioniza-
ica Acta 702 (2011) 1– 15 13
tion mechanisms are similar to that employed in conventional APPI.DAPPI has the potential to characterize both polar (e.g., verapamil)and nonpolar (e.g., anthracene) compounds from a range of sur-faces. It has also been used for the direct characterization of analytesin food and environmental samples and for the screening of illicitdrugs [182–185].
5. Conclusion
The extensive array of newly developed ambient ionizationtechniques described herein allow the rapid, online, real-time,and high-throughput analysis of samples in their native condi-tions and in the open air without sample pretreatment; they havesparked significant interest and new applications in various fieldsof academic or industrial interest. These techniques employ vari-ous methods for the sampling of solids, liquids, and gases throughdesorption and ionization processes; they are facilitating the devel-opment of a growing number of powerful analytical methods witha broad range of applications in areas such as pharmaceuticalanalysis, food safety, reaction monitoring, environmental analy-sis, biological imaging, proteomics, metabolomics, forensics, andexplosives detection for antiterrorism. In this tutorial paper, wehave logically and methodically sorted the large family of ambientionization techniques—with their different sampling designs andcombinations using ESI, APCI, or other ionization techniques—intothree main categories: (i) direct ionization (directly ionizing ana-lyte in liquid in an electric field), (ii) direct DI (charged reactivespecies or metastable atoms generated by an ambient ionizationsource are brought to the sample surface for desorption and ion-ization), and (iii) two-step ionization (analyte molecules are firstformed through laser irradiation, shockwave treatment, thermalenergy, or nebulization and then transferred to the ambient ion-ization source for post-ionization). Although ambient ionizationtechniques have become an important part of modern MS, muchremains to be learned about the fundamental mechanisms andproperties underlying many, if not all, of these methods. The discov-ery of new applications and the optimization of existing ambientionization techniques will surely benefit from such pursuits.
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