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Desorption electrospray ionization and other ambient ionization methods: current progress and preview
Demian R. Ifa,a Chunping Wu,a Zheng Ouyangbc and R. Graham Cooks*ac
Received 1st December 2009, Accepted 16th February 2010 First published as an Advance Article on the web 2nd March 2010 DOI: 10.1039/b925257f
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Mass spectrometry allows rapid chemical analysis of untreated samples in the ambient environment. This is a result of recent rapid progress in ambient ionization techniques. The most widely studied of these new methods, desorption electrospray ionization (DESI), uses fast-moving solvent droplets to extract analytes from surfaces and propel the resulting secondary microdroplets towards the mass analyzer. This review of DESI and other ambient methods centers on the accompanying chemical processes. Manipulation of the chemistry accompanying ambient ionization can be used to optimize chemical analysis, including molecular imaging. Solvent effects, geometry effects, electrochemical processes and mechanisms are covered. Extensions of the methodology to solution-phase analysis, to stand-off detection and to therapeutic drug analysis using miniature mass spectrometers are also treated.

1. Introduction and summary of current status

Analytical characteristics In 2004 DESI (Fig. 1), the rst of the now almost 30 ambient ionization methods for mass spectrometry, was reported.1,2 These methods and the current state of the subject of ambient ionization are described in some detail in the accompanying review by Weston.3 The present review/preview concentrates on emerging topics, especially those judged likely to have most
Fig. 1 Illustration of desorption electrospray ionization (DESI).1 Department of Chemistry, Purdue University, West Lafayette, Indiana 47907, USA. E-mail: cooks@purdue.edu b Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana 47907, USA c Center for Analytical Instrumentation Development, Purdue University, West Lafayette, Indiana 47907, USA This paper is part of an Analyst themed issue on Ambient Mass Spectrometry, with guest editors Xinrong Zhang and Zheng Ouyang.
a

impact on the future direction and applications of ambient ionization. A working denition of ambient ionization is that ionization occurs externally to the mass spectrometer and that analyte ions, not the entire sample, are introduced into the mass spectrometer.

Demian Ifa received his B.S. in pharmacy from the State University of Sa o Paulo (UNESP), Brazil. He received his M.S. in organic chemistry from the University of Rio de Janeiro (UFRJ) and his Ph.D. in pharmacology from the University of Sa o Paulo (USP), Brazil. He is an associate research scientist at the Aston Labs for Mass Spectrometry at Purdue University working on Demian Ifa the development of desorption electrospray ionization and its applications to imaging and quantitation.
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Chunping Wu

Chunping Wu received her M.S. in analytical chemistry in 2005 from University of Illinois at Chicago. She is currently pursuing her Ph.D. degree at Purdue University-West Lafayette under the direction of Prof. R. Graham Cooks. Her research focuses on practical applications and fundamentals of ambient ionization techniques. She is soon to take up a position in the Analytical Science Lab of ExxonMobil Research & Engineering Co. at Annandale, New Jersey.

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(Note that electrospray ionization (ESI), atmospheric pressure MALDI, and atmospheric pressure chemical ionization (APCI) are excluded by this denition.) Ambient ionization methods, it follows, allow the ionization of untreated samples in the open environment. There is no requirement for sample preparation and as a result, analysis is rapid with the time scale being governed by the time needed to present the sample to the mass spectrometer. High throughput is a direct consequence of these features. Internal energy distribution of ions produced in DESI is around 2 eV,4 which is similar to that in ESI, so limited fragmentation occurs in DESI or in other ambient ionization methods. DESI is characterized by: High speed and throughput total analysis speed typically less than 5 s because of the lack of sample preparation; this allows high-throughput analysis. Soft ionization very little molecular fragmentation occurs, making it easier to identify compounds in mixtures. Molecular specicity particular compounds are characterized by their molecular weights as read from the mass spectrum; this information is quickly and easily enhanced using MS/MS data. Positive and negative ionization ions of either polarity are formed, increasing the range of application and versatility of the method. High sensitivity like many ambient ionization methods, DESI displays excellent sensitivity with absolute detection limits for pure compounds often in the sub-nanogram range. Low matrix and salt sensitivity it was been demonstrated5 that DESI is much less sensitive to salt effects than is ESI, minimizing or eliminating the clean-up required for biological samples. Low substrate/surface requirements there is no special requirement for the surface from which analytes are examined although rough insulating surfaces work particularly well. Universal applicability compounds of virtually all classes from hydrocarbons to highly functionalized compounds can be ionized. The method not only applies to all small molecules (mol. weight < 1000 Da) but also has success with proteins. Quantitative accuracy and precision is controlled by the nature of the internal standard and performance data and is

similar to that of ESI when the standard can be mixed into the sample. For some materials this is not possible and then only semi-quantitative data are obtainable. A set of interrelated methods A large family of ambient ionization methods has been described,68 all sharing to various extents the properties just outlined. They divide readily into two main classes: those like DESI which depend on a solvent spray, and others like direct analysis in real time (DART)9 which utilize plasmas to create gasphase ions. The former are ESI-like, the latter APCI-like. In addition, a number of methods achieve the two steps of desorption and ionization by means of two separate agents, e.g. ELDI10 and LAESI11 both use lasers for desorption followed by electrospray ionization of the desorbed neutrals. There are many variants on methods, depending for example in the plasma methods on the type of discharge, the power and the geometry. An early method was plasma assisted desorption ionization (PADI);12 a related simple plasma method, low temperature plasma (LTP)13 (Fig. 2), allows the plasma to interact directly with the sample and creates ions that are transferred by gas ow and vacuum suction into the mass spectrometer. There has been a tendency to name new methods based on minor differences to existing procedures and there is a need for rationalization of nomenclature.

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Fig. 2 Illustration of low temperature plasma (LTP) probe for desorption and ionization.13

Zheng Ouyang received his B.E. and M.E. degrees in automatic control from Tsinghua University, Beijing, China, his M.S. degree in physical chemistry from West Virginia University, Morgantown, and his Ph.D. degree in analytical chemistry from Purdue University, West Lafayette, IN. He is an assistant professor with the Weldon School of Biomedical Engineering at Purdue University. Zheng Ouyang His research interests include the development of miniature mass spectrometry analysis systems and their applications for biomedical analysis.
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Graham Cooks received Ph.D. degrees from the University of Natal (now KwaZulu-Natal) and Cambridge University. His interests involve instrumentation, fundamentals and applications of mass spectrometry.

Graham Cooks

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The plasma methods have the advantage relative to the spray methods of not requiring expendables (a low carrier gas ow, even air, sufces). They give mass spectra for low molecular weight compounds that are similar to DESI spectra and the experiment is sensitive, soft and rapid. Some compounds work less well in LTP than in DESI and it is a characteristic of all the plasma methods that ionization is increased on heating the sample. This is an indication of the relationship to APCI. A good example is the case of melamine, the LTP ionization of which is discussed elsewhere14 and also in this issue.29 Although the ambient ionization methods are normally applied to solids, they can be used to examine surfaces of solutions too (see Section 6, below) and a case can be made that the vapor-phase getting of molecules by charged microdroplets15,16 rstenau, represents a vapor-phase rst described by Fenn and Fu ambient ionization experiment.

fundamental process.20 Note charged droplet spray, which characterizes DESI, can be produced without application of an external voltage, a simplied but less efcient procedure.21

3. Solvent, substrate and geometrical effects

Solvent effects Solvent choice greatly affects the DESI ionization efciency, an expected result given the key role of dissolution in the mechanism. Much follows: the fact that methanol/water is a standard solvent for many polar molecules, both in the positive and negative ion modes; the fact that addition of small amounts of acid favors positive ion formation; the fact that a correlation exists between the solubility of a compound in a particular solvent and the success of that solvent in DESI.22 The insights into the DESI mechanism obtained from dynamical simulations19 suggest that surface-active analytes should be more efciently sampled in the splashing process which creates secondary microdroplets. This is consistent with the observation that the addition of surfactants to the DESI spray solution gives enhanced instantaneous currents, hence improving the detection limits.22 The effect is ascribed at least in part to a reduction in surface tension caused by the presence of the surfactant. Surfactin and several common industrial surfactants, characterized by very high surface activities, display this enhancement when added in small concentrations to normal DESI spray solvents.22 Preliminary results indicate that surfactants can effectively increase the sensitivity for the analysis of food chemicals, explosives, and pharmaceuticals. The basis for the success of the ambient spray methods is the dissolution of analytes in the microdroplets or in a thin lm of solvent. It seems likely that the charged nature of the sprayed droplets plays a role in the speed of dissolution and hence in the effectiveness of the process. It is also of great interest that the nature of the spray solvent, including any added solute, might play an important role in modifying the analyte and so allowing its successful analysis. Such a modication might involve chemical derivatization (reactive DESI, below) or the solvent might be

2. Key mechanistic features

DESI The main DESI mechanism has been described as droplet pickup.17 This might occur in a single step but in most experiments it appears to involve initial wetting of the surface to dissolve the analyte, followed by splashing on the arrival of subsequent droplets with emission of secondary microdroplets. Evidence for this mechanism comes from simulations and from experiments on the velocity and diameter of the droplets as determined by phase Doppler particle analysis. The average droplet velocity is about 150 m/s and the average diameter of the droplet is about 3 mm (Fig. 3A).18 The internal energy distribution of ions produced in DESI, determined using the survival ion yield with thermometer ions, is comparable to ESI with a median value around 2 eV (Fig. 3B).4 This is a low internal energy and explains the limited amount of fragmentation seen in the mass spectra. Simulations of the DESI process show the formation of dozens of microdroplets resulting from a single dropletthin lm collision event (Fig. 3C).19 Experiments in which DESI spectra are recorded at 50 Hz might speak to the short time scale of the

Fig. 3 (A) Droplet velocity and diameter measurement of a 5 mL/min water spray using a 200 psi N2 inlet pressure. (B) The breakdown curve obtained for the six thermometer ions and the internal energy distribution as functions of the critical energy. (C) DESI simulation showing the side view and topdown view of contours of the indicator function at four simulation time steps with an incident angle a 55 . Adapted from refs 4, 18 and 19.

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chosen to hydrolyze, saponify, or otherwise chemically modify a sample to effect an increase in ionization efciency.

Non-proximate (stand-off) detection Whatever geometry is used, the desorbed ions are transferred to the mass spectrometer by an ambient pressure sample transfer line. Ion transportation through this line at ambient pressure is the source of signicant inefciencies in DESI and LTP, and presumably in the other ambient ionization methods. Early stand-off studies using DESI on a lab-scale ion trap mass spectrometer were successful in recording high-quality spectra over distances of 13 m.28 These experiments simply used the mass spectrometer suction to transport analyte but this is not efcient and the loss in signal was ca 102103, over this range of distances. (Remarkably, because most of the noise was chemical noise associated with background or contaminant species and because these were more easily lost on transport than the analyte ions, the S/N ratio actually increased with distance.) Efciency can be increased by using a large capacity, low speed supplementary pump to move large volumes of gas to the MS inlet under laminar ow conditions, then intercepting the ions. These conditions allowed 8 m transfer with the lab-scale mass spectrometer (unpublished data). They were also essential to effective transport of ions into the Mini 10 handheld mass spectrometer, for example from the LTP probe (Fig. 5).29 The increasing efciency of transport of ions generated by DESI and by LTP into the miniature instrument, in situ analysis with stand-off detection seems likely to be achievable. This would mean the simultaneous achievement of stand-off detection with high speed, high sensitivity and high molecular specicity for trace analytes in complex matrices.

Substrate effects Various substrates (stainless steel, gold, glass, ceramic and polymers) show surface-charging effects which depend on their conductivity. Surface charge distributions on insulating substrates build up quickly (ca. 10 s) after initiation of the spray and then remain constant for long periods (minutes) as measured using a capacitive probe.23 The region closest to the sprayer tip has the highest charge density, and the charge density gradually decreases as the distance to the sprayer tip increases. Even on highly insulating surfaces, these effects do not preclude recording of high-quality mass spectra, as similar effects do in secondary ion mass spectrometry, presumably because the high momentum of the arriving charged droplets makes them impervious to coulombic effects.

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Geometry effects There are geometrical requirements for the spray methods specically DESI which are the result of the macroscopic masses and momenta of the arriving and leaving droplets and the directed gas stream in which the droplet/surface collisions take place. Once optimized, these angular requirements are stable for long periods but the shallow take-off angle has proven problematic to some investigators. Two alternatives have been suggested. One involves so-called geometry independent (GI)DESI,24 Fig. 4, in which both the incident and emergent spray angles are xed and near-normal to the substrate, so no geometry optimization is needed. This GI-DESI experiment can be thought of as using reection geometry. The alternative method uses transmission mode geometry. In this experiment, the sample is present on a mesh and the secondary droplets continue in the same direction as the primaries, carrying analyte into the mass spectrometer.25,26 In spite of the reduced sensitivity of the transmission mode (Fig. 4C), Brodbelt and co-workers27 have made effective use of this geometry, including experiments in which surface reactions generate easily ionizable functional groups so as to increase specicity and sensitivity (compare reactive DESI, discussed below).

4. Chemical reactions
Reactive DESI The ambient ionization methods, especially the spray methods like DESI, involve conditions (temperature, pressure, solvent system, etc.) that are remarkably similar to those encountered in ordinary solution-phase chemical reactions. It is therefore natural to draw on established solution-phase chemistry and to add reagents to the spray solvent or use gas-phase reagents to enhance performance. Reactive DESI, as this experiment is called, was rst used to increase the specicity of ionization of particular analytes. Examples are given in Fig. 6, and attention is

Fig. 4 Two simplied geometries for DESI. (A) and (B) Reection mode (geometry independent) GI-DESI and its use in high-throughput analysis of metabolites in a bacterial matrix on a 96-well plate.24 (C) Transmission mode (TM-DESI).27

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Fig. 5 Diagram showing the use of a supplemental suction to assist in transfer of ions from an LTP ambient ion source to the mass spectrometer.29

drawn to the relatively non-polar analytes like cholesterol, fat-soluble vitamins (A and D), and anabolic steroids for which DESI is normally less sensitive. Reactive forms of the plasmaand laser-based ambient ionization methods have also begun to be explored. The potential value of this type of study can be seen by considering the DESI-MS spectrum of a tissue section which shows no signal for cholesterol using methanol/water spray reagent, but a strong signal for the cholesterol derivative when betaine aldehyde is included as a reagent in the spray solvent.30

As opposed to simple cationization31 or anion addition,32 redox reactions,33 hostguest complex formation3436 and chemical bond-forming reactions30,3740 have gained increasing attention in reactive DESI experiments since they are analogous to ordinary solution-phase functional group reactions. Other reaction types are also of interest in ambient ionization. For example, charge exchange between an ionized vapor-phase compound such as toluene and a physisorbed analyte molecule is the basis for the ambient method known as desorption atmospheric pressure chemical ionization (DAPCI).41

Fig. 6 Examples of reactions used in reactive DESI experiments to improve sensitivity of detecting cholesterol,30 anabolic steroids,37 cis-diols,38 phosphonate esters,39 and cyclic acetals.40

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Chemistry in evaporating droplets It is remarkable that the cholesterol derivatization and other reactions can occur in the short time available in a DESI imaging experiment (approximately 1 s per pixel). The reactions which have been studied so far (Fig. 6) all occur to a signicant extent on this time scale. Several questions arise: (i) what are the rates of these reactions? (expressed differently, what fraction of the analyte is converted to products on the DESI time frame?); (ii) what is the role of charge in driving these reactions?; (iii) what is the role of the solvent in these reactions?; (iv) what are the roles of droplet size and ionic strength?; and (v) what ancillary means are available to affect reaction rates? It seems likely that the high (or low, depending whether the voltage applied is for negative or positive ion detection) pH associated with evaporating droplets drives reactions must faster than they would go under normal conditions. These important considerations are under active investigation.

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Electrochemistry As is also the case with electrospray ionization, electrochemical processes are inherent in DESI. However, in contrast to ESI, the additional electrode (substrate used in DESI) acts as a DC capacitor, and electrochemical redox reactions can occur on this surface.42 There is an asymmetry between the positive and negative ESI modes with oxidation in the positive ion mode being much more common, although reduction is observed in

some cases. By contrast, reduction in DESI seems to be extremely rare.43 During operation under standard conditions, there are few cases in which electrochemical processes signicantly inuence the DESI mass spectra of organic compounds. One such case is that of easily oxidized or reduced compounds, e.g. benzoquinone (Fig. 7A). Based on the electrosonic spray ionization (ESSI) behavior in the negative ion mode, it was expected that 1,4-benzoquinone would be reduced in DESI to form 1,4hydroquinone and then be ionized to yield the [M H] evenelectron ion of m/z 109. Instead, as can be seen in Fig. 7A, an odd-electron radical anion of benzoquinone is observed. This result was conrmed by performing the same experiment with labeled 1,4-benzoquinone-d4. This difference in ESSI/DESI behavior is obviously associated with the presence of the additional electrode (the substrate used in DESI, where electrochemical redox reactions can occur). It is likely that dischargeinduced electron attachment (see next section) is responsible. The combination of electrochemistry (EC) and mass spectrometry (MS) is a powerful analytical tool to study and utilize redox reactions. Previously, EC/MS coupling was realized using such ionization methods as thermospray (TS),44 fast atom bombardment (FAB)45 and particularly electrospray ionization (ESI).46 Recently, on-line coupling of EC with DESI-MS has been demonstrated.47,48 As a result of the direct sampling nature of DESI, several useful features of such a combination have been found, including the simple instrumentation, rapid response time (e.g. 3.6 s in the case of dopamine oxidation), freedom to choose the more favorable ionization mode of DESI and traditional electrolysis solvent systems, and the absence of background signal possibly resulting from ionization occurring when the cell is off. More importantly, using this new coupling apparatus, three disulde bonds of insulin were fully cleaved by electrolytic reduction and both the A and B chains of the protein were successfully detected on-line by DESI-MS (Fig. 8).

Electrical discharge-induced oxidation Besides redox reactions associated with electrochemical processes which are inherent to DESI, discharge-induced oxidation occurs for specic congurations when DESI is performed in air, i.e. discharge can occur between the emitter tip and inlet capillary of the mass spectrometer, if sufciently closely positioned (ca. 1 mm). The occurrence of unintended oxidation in DESI was rst noted by Van Berkel and co-workers.49 These oxidation processes can be advantageous as a means of in situ derivatization, for example, for hydrocarbon analysis. Such experiments effectively combine plasma ambient ionization with spray ionization (LTP and DESI). A good example is provided by the analysis of condensed-phase saturated hydrocarbons which are difcult to ionize by many other methods.50 By spraying with methanol/water containing a reagent that reacts with alcohols and by doing so in the presence of a discharge in air which generates hydroxyl radicals, the alkane is converted to the alcohol and then derivatized in situ to give an easily ionized product. Fig. 7B illustrates the success of this readily performed reaction. It shows that non-functionized saturated hydrocarbons can be oxidized in DESI, with multiple oxidation and dehydrogenation steps, to generate ketones and alcohols.
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Fig. 7 (A) Electrochemical reduction of 1,4-benzoquinone (BQ) under DESI conditions, as compared to ESSI. (B) Reactive DESI of n-octadecane (M) with deliberate discharge-induced oxidation to generate the alcohol which is reacted in situ with betaine aldehyde (BA). Adapted from refs 43 and 50.

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endogenous chemicals (inks, drugs of abuse, pharmaceutical molecules, or biological molecules) in various objects (ngerprints,52 documents,53 tissue sections,30,5458 etc.). Because of the ambient nature and softness of DESI ionization, the unmodied native sample can be examined for information on chemical distributions. MS imaging of tissue sections with DESI is carried out under ambient conditions, without sample pretreatment (other than sectioning). This avoids any possibility of contamination with exogenous compounds. DESI tissue imaging to recognize diseased tissue for tumor diagnostics is an ongoing interest.55 As shown in Fig. 9, some specic phospholipids have higher signal intensities in the tumor as compared to the normal tissues, so can be used as markers for tumor diagnostics. The diagnostics using sample-preparation-free DESI imaging (data acquisition time ca. 1 h) are well within the limited bounds now available with diagnostic results from histopathology.

Reactive imaging The combination of ambient ionization with specic solutionphase reactions which take place during the imaging experiment has already been introduced in the discussion of reactive DESI. With reactive DESI in an imaging mode, naturally occurring cholesterol in rat brain tissue (ca. 13 mg/g) is easily imaged under ambient conditions. A full 2-D image at 200 mm resolution can be recorded within an hour (1 s per pixel). The ion image of the peak at m/z 488.5 (corresponding to [BA + Chol]+), extracted from the full set of data, shows the expected increase in cholesterol levels in the white matter (e.g. corpus callosum, anterior part of anterior commissure, cerebellum) as compared to the gray matter of rat brain.30 The spatial distribution of cholesterol mapped by DESI is consistent with literature results and this study serves to emphasize the potential value of this imaging mass spectrometry experiment in the biological sciences. Lipids are not the only endogenous compounds that can be imaged by the ambient ionization methods. LAESI has been found to be suited to protein imaging11 and it has also be used to produce 3-D images.59 The low concentrations of endogenous hormones (testosterone, androstenedione, estradiol, etc.) in biological uids and tissues make the direct detection and quantication of such hormones using DESI challenging, although there are exceptions like the adrenal hormones.60 Even with reactions shown above, the direct analysis of low concentrations of hormones in biological uids (whole blood, plasma, or serum) could not be achieved. New chemical reactions with higher reaction efciency will be established to improve sensitivity to allow their detection. Another restriction is that lipids with relatively low proton afnities or cation afnities could not be efciently ionized, and their signals are suppressed by other lipids. For example, the signal of phosphatidylethanolamines (PEs), with lower ionization efciency than phosphocholines (PCs), is often suppressed by PCs in the positive ion mode of DESI. Reagents are needed to specically react with PE to enhance their signals. On the other hand, reactions can also be implemented to selectively ionize members of a specic class of polar lipids in applications where differentiation of compound classes is needed.
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Fig. 8 (+)-DESI-MS spectra acquired when a solution of insulin (0.1 mM) in H2O/CH3OH (1:1 by volume) containing 1% acetic acid owed through the thin-layer electrochemical cell with an applied potential of (a) 0.0 V and (b) 1.5 V. The inset in (a) shows the structure of intact insulin which contains three disulde bonds.

The resulting alcohols are selectively detected using reactive DESI with betaine aldehyde as the reagent. The two-step in situ derivatization in DESI can be utilized to analyze non-polar petroleum samples under ambient conditions, and to provide accurate carbon distributions and exact mass measurements when mass analysis is done using a high mass resolution orbitrap. However, the discharge in DESI cannot be controlled accurately enough to adjust the extent of oxidation. Controllable electrical discharges in the vicinity of the microdroplets might help to create reactive species to react with the analytes, in order to enhance the sensitivity of detecting compounds with low ionization efciency. The separate electrical discharge might take the form of a low temperature plasma (LTP),13 in which the amount of reactive species generated in the air can be controlled by adjusting the current and gas ows. By combining DESI and LTP, oxidation (or other reactions) might be useful in the analysis of lipids as well as hydrocarbons and steroids.

5. Application areas
The companion review by Weston3 includes extensive coverage of many applications of the ambient ionization methods. For that reason, this section focuses on aspects of just two topics: imaging and in situ analysis. Imaging A signature application of DESI is mass spectrometric (MS) imaging,51 used to map the spatial distributions of exogenous or
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Fig. 9 Negative ion mode tissue imaging of canine bladder tissues including areas of cancer and adjacent normal tissues. H&E-stained tissue sections of the tumor tissue and the tissue adjacent to the tumor were shown on the left panel. Ion images of PS(18:0:18:1) at m/z 788.6, PI(16:0/18:1) at m/z 835.7, and PI(18:0/18:1) at m/z 863.7, indicate that these lipids are more enriched in the tumor as compared to the normal tissues. Adapted from ref. 55.

MS/MS imaging Tandem mass spectrometry provides additional specicity for many ambient ionization experiments, chemical specicity that is often lacking in the simple mass spectra given by pure compounds and in the very complex spectra given by biological tissue sample. Lipids in particular can occur in isomeric and isobaric (same nominal mass) forms and additional information from mass spectrometry is desirable to conrm assignments. The potential value of MS/MS imaging for this purpose is apparent from studies of chemical imaging of latent ngerprints.52 Natural sebaceous oils left as ngerprints can be detected and identied (Fig. 10A). This observation can be extended to the identication of illicit drugs, explosives, pharmaceutical compounds and other chemicals, which have been handled through the analysis of latent ngerprints, powdered prints, and tape-lifted prints on a variety of types of surfaces. As shown in Fig. 10, MS/MS images have similar quality in these cases to MS images and they do not take signicantly longer to record for targeted analytes, but they provide greatly increased chemical specicity. High mass resolution imaging An approach to improved molecular imaging that can supplement or replace MS/MS lipid imaging is high mass resolution imaging. The fact that isobaric species may only be present in isolated regions of the tissue means that a tissue section might need to be exhaustively analyzed (i.e. imaged) by recording MS/ MS data for each peak to identify isobaric species, a timeconsuming procedure involving many MS/MS scans per pixel. Imaging using the high resolution but relatively rapid Orbitrap mass spectrometer solves this problem. The improved resolution of the instrument compared with an ion trap resolves most isobaric species and increases the amount of chemical information obtained from complex samples.
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An example of the value of DESI-MS imaging using a commercial LTQ/Orbitrap XL mass spectrometer, in distinguishing isobaric species in the mouse brain (with mass resolution of 30 000), is shown in Fig. 11. In these experiments, the chemical images of phospholipids were reconstructed using BioMap (freeware, www.maldi-msi.org) and it reveals their specic distributions in substructures of the brain. Note that the mass difference (0.065 amu) between two species, A2 and B1, requires a resolution of 15 000 for their resolution. Coupling DESI to hybrid instruments such as ion mobility/mass spectrometry would also increase specicity. These experiments have been proposed;61 however, its application to imaging has not been exploited.

Increased spatial resolution in imaging The spatial resolution of the laser-based ambient ionization methods potentially can be high, even with femtosecond lasers very high. At present the state of the art for LAESI is on the order of 100 mm, and most DESI work is done at 180220 mm, although much lower values have been reported.62 A key requirement in microprobe imaging is to use the minimum resolution needed simply because time required for an experiment increases with the inverse square of the spot size. One solution to this, as in the MALDI work of Heeren and co-workers,63 is to use the microscope rather than the microprobe imaging mode but the complexity of the instrumentation is much greater. Another (partial) solution is to examine restricted regions at high spatial resolution (as is commonly done in very high resolution SIMS experiments).64,65 The examination of small local areas can be done with ne needles, which are used to remove material that can then be examined in the open environment66 or an atomically sharp needle can be used to provide a local site of high eld and hence favored desorption. Note that not all these experiments are amenable to ambient ionization.
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is the standard but slow method, or (ii) using a 2-D array of sources to cover the area, or (iii) by a compromise in which a smaller number of sources is used to sample the surface, for example by performing a line scan using a 1-D array of ion sources across the area to be studied. This might be the best approach given the constraints of the systems involved. The relatively large volume of solvent required by DESI (15 mL/min) makes the 2-D approach impractical. However, a linear array of ion sources which also allows rapid sampling has proven to be useful in both the low temperature plasma experiment as well as the DESI experiment.

In situ trace analysis Particular interest attaches to performing organic trace analysis in situ for a variety of public safety applications. This requires a portable mass spectrometer and the simultaneous achievement of a set of performance characteristics that are highly demanding. The principal ones are as follows:  Hand-portable mass spectrometer which is rugged, reliable and autonomous.  Automated data interpretation and library comparison.  Minimal or no sample preparation/pretreatment.  High selectivity and sensitivity.  Detection and quantication in complex matrices.  Total analysis time of seconds.  High-throughput capabilities.  Non-proximate (stand-off) detection.  Large area detection. A variety of hand-portable mass spectrometers have been described and several have been commercialized.67 Our own efforts have focused on ion trap instruments, because of the simplicity of access to MS/MS capabilities and their operation at much higher pressures than any other type of mass spectrometer.

Fig. 10 Virtual DESI image of the fatty acid cis-hexadec-6-enoic acid (m/z 253) from a LFP blotted on glass (A) and lifted by an adhesive tape (B); D9-THC and/or cannabidiol on paper as identied by the MS/MS transition m/z 313/245 (C); D9-THC distinguished from cannabidiol by the unique MS/MS transition m/z 313/191 (D). Adapted from ref. 52.

Large area detection Small area analysis represents one frontier in ambient ionization, large area analysis another. Large area detection has been pursued to efciently survey larger samples using two types of ionization methods: DESI and LTP. The analysis of large areas by intrinsically small area sources (DESI and LTP) can be achieved in three ways: (i) rastering the source across the area, which

Fig. 11 DESI-MS imaging of a coronal section of mouse brain in the negative ion mode (optical reference in blue, Nissl stained). Compound A is distributed on the whole section of the brain as observed by mapping the isotopic series of ions A1 (m/z 885.544), A2 (m/z 886.542) and A3 (m/z 887.551). Another compound, B, is distributed only in the central areas of the brain such as corpus callosum, thalamus and the caudoputamen as observed by mapping the isotopic series B1 (m/z 886.607) and B2 (m/z 887.605).

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The miniature mass spectrometer was initially built and designed with positive ion mode analysis only. More recently, capabilities for negative ion detection were added.68 Negative ion detection using the portable mass spectrometer further improves both the specicity and the sensitivity of detection. Implementation of negative ion detection on the miniature mass spectrometer represented a challenge given that high voltage conversion dynodes had to be incorporated. Good negative ion mode DESI and LTP are now available. From the efforts to improve negative ion detection, it is now possible to achieve using DESI: (a) interrogation of larger areas (ca. 6 cm2), (b) increased specicity and sensitivity using negative ion detection, (c) rapid analysis (< 30 s) and (d) low detection limits of 1 mg/cm2 for randomly spotted large area samples. Most of the portable mass spectrometers described in the literature are not capable of desorption or spray ionization. A key to coupling atmospheric pressure ionization sources to miniature mass spectrometers (which have very limited pumping capabilities) is a discontinuous atmospheric pressure interface (DAPI).69 This interface is open during the ionization part of the scan period to let ions (and air) enter the ion trap, and then closed for the remainder of the scan period to maintain the low working pressure of the mass analyzer. Thus, the ow conductance and opening time of the interface was adjusted so as to optimize the number of ions entering the ion trap. The air is pumped away during the closed time while the ions remain trapped and they are then analyzed once the pressure falls back into the appropriate range. The method successfully trades slower analysis speed with the use of ambient ionization coupled to a miniature mass spectrometer.70 The combination of miniature mass spectrometers and the DESI source has great analytical capabilities and potential, especially for in situ analysis. In a typical demonstration experiment, DESI was used with a Mini 10 mass spectrometer to detect 25 ng cocaine on a Teon surface.

Fig. 12 DESI analysis of liquid samples.47

This is consistent with our understanding of the mechanism of DESI, since the required dissolution of the analyte is already achieved. One of the additional features of solution-phase DESI is that it is easy to desorb large proteins directly from the solution (Fig. 13).47 Also, using an organic solvent like methanol or acetonitrile as the spray solvent, it is possible to perform on-line desalting for high salt-containing biouid samples such as urine.47,72 Fig. 14 shows the successful detection of a trace amount of methamphetamine (MA; 1 mg/mL, a drug of abuse) in raw urine while direct ionization of the same sample by ESI failed to produce observable signal (i.e. protonated methamphetamine of m/z 150).47 Furthermore, ion/ion reactions under ambient conditions can be carried out in the liquid DESI experiment. For instance, ion/ion reactions of doubly charged Zn(II) complex ions were used to selectively bind negatively charged phosphoserine in the presence of serine.47 The use of plasma methods to sample the surfaces of liquids is also well-established. For instance, photochemicals curcuminoids were successfully detected in commercially available functional beverages containing turmeric extract and curry powder by DART-MS.73 The rst reports on atrazine determination in solution were described in the rst publication on LTP. Therapeutic drug analysis from serum

6. New methodologies and improved performance

Liquid DESI Ambient ionization, the family of techniques in which samples are analyzed in their native state in the ambient environment, has been performed almost exclusively on solid samples. The advantages of the methodology are obvious, including high throughput and lack of contamination of the vacuum system of the mass spectrometer because the sample as a whole is not introduced into vacuum. The numerous DESI and DART studies reported in the literature have been done on solid samples, with the exception of some recent DESI studies on solutions from the labs of Zhang71 and Chen.47 In the research of Zhang and co-workers, solutions containing analytes in multiple capillaries are driven out by the DESI nebulizing gas and sampled in a high-throughput fashion for MS analysis. In the work of Chen and Miao, the solution is sheeted on a surface and then the normal DESI spray is performed (Fig. 12). This experiment gives excellent results, not only in terms of sensitivity and high-quality MS and MS/MS spectra, but it appears to give superior performance to normal solid DESI in terms of the molecular weight range of the compounds that can be studied.
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New methods for quantitation of drugs in biological matrices which reduce the need for sample preparation are being developed using such direct analysis methods as desorption

Fig. 13 MS spectra showing the direct DESI-MS analysis of solutions containing bovine serum albumin (BSA).47 The insets show the corresponding deconvoluted spectra.

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Fig. 14 MS spectrum showing the DESI-MS detection of methamphetamine (1 mg/mL) in a raw urine solution.47 DESI spray solvent used was methanol/acetic acid (1:0.03 by volume).

paper dried blood spots. This method has the advantages of cost, stability and convenience that are compelling. Until recently, the analytical measurements of drugs in blood associated with these dried blood spots involved standard methods of extraction followed by liquid chromatography-MS/MS. This has now begun to change with direct, on-paper DESI analysis.75 The main concern is the addition of internal standard; it can be added to the extract after complete extraction or to the whole blood before addition to the paper. Methodology in which it is incorporated into the porous medium for controlled elution is being explored.76,77

Paper spray electrospray ionization (DESI).74 The endpoint of these experiments is the use of small, low-cost mass spectrometers that are more cost-effective in a clinical point-of-care setting. One such experiment examined the anticancer drug imatinib, the current frontline treatment for chronic myeloid leukemia (CML). Although very efcacious in most cases, imatinib pharmacokinetics shows signicant inter-patient variability. Also, there is an established relationship between imatinib exposure and the drugs efcacy and toxicity. As a result, some patients will not respond or will show unusually severe side-effects when given a standard dose of imatinib. Fig. 15 shows a calibration curve for imatinib in raw, untreated serum analyzed directly by DESI-MS.74 In this experiment, imatinib was spiked into serum at varying concentrations. The drug-spiked serum was spotted on a micro TLC plate and analyzed immediately by DESI-MS without any separation. The TLC plate gives improved sensitivity due to increased surface area for extraction. The results indicate a promising future for therapeutic drug monitoring (TDM) in a clinical setting. The development of rapid quantication using miniature mass spectrometers that are robust and allow rapid automated sample preparation methods is a next requirement. This achievement could lead to the development of a device for accurate measurement of levels of drugs in blood at patients bedsides in a matter of minutes. Dried blood spot analysis There has been a dramatic increase in interest in the pharmaceutical industry in storing and transporting blood as spots on An alternative to DESI for the analysis of dried blood spots is paper spray,76 a technique with close connections to DESI and also to nanoelectrospray. In this experiment, the paper containing the blood spot (or other biological uid) is wetted with solvent (methanol/water for example) and a high voltage connection is made. The mixture of solvent and analytes in the blood is ionized by a spray ionization method. Paper spray is a very simple, robust and user-friendly ambient ionization method. Typically the paper substrate is cut into the shape of a triangle (so as to have a macroscopically ne point). It is believed that the spray process is very similar to that in an array of nanospray emitters that the paper serves to lter cells, that it can have chromatographic and electrophoretic separation effects, and that the point in the paper speeds up the wick-driven solvent ow just as is done when a river channel narrows. Below are depictions of the paper spray experiment and the analysis of angiotensin in the positive ion mode (Fig. 16). This ionization method is suitable for low molecular weight organic compounds as well as biomolecules, including peptides and proteins. There are numerous other uses of this ionization method which are still to be explored. One that is under exploration is the use of the porous material as a surface wipe and then as the substrate for mass spectrometry.

7. Concluding comments
The characteristics of the ambient ionization methods make them particularly well-suited to the study of dynamical systems. The absence of sample preparation and the immediate responses ensure this t. One example is the characterization of products and intermediates in reacting chemical systems, an experiment that can be done by taking aliquots of the reacting solution and examining them after drying on a suitable substrate or, even more directly, by examining the surface of the reaction mixture.72 The sine qua non application of ambient mass spectrometry must be in vivo analysis, especially intrasurgical analysis. Signicant steps towards achieving this objective are being taken. In one application, Agar and co-workers use DESI in the surgical suite in parallel with standard histochemical examinations during glioma surgery (personal communication). In another application, Schafer et al.78 use APCI to sample the surgical smoke created during electro-cutting and are able to draw conclusions regarding the biological state of the tissue accessed at any time from the mass spectra recorded.
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Fig. 15 Calibration curve for imatinib in raw, untreated serum.74

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Fig. 16 Paper spray (A) experimental setup, (B) samples spray resulting from application of high voltage potential, and (C) positive ionization of angiotensin I solution. Panels (B) and (C) modied from ref. 76 with permission from John Wiley & Sons, Inc.

Acknowledgements
Support is acknowledged from the National Science Foundation (NSF 0848650), the Department of Homeland Security (DHS 08116108) and the Ofce of Naval Research (N00014-05-1-0405) for support. We also thank Thermo Scientic Instruments, Inc. and ICx, Inc. for support and valuable interactions. Helpful comments by Dr Daniel Weston (AstraZeneca R&D Charnwood, UK) and Hao Chen (Ohio University) are greatly appreciated.

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