desorption electrospray ionization and other ambient ionization methods: current progress and...

7/27/2019 Desorption electrospray ionization and other ambient ionization methods: current progress and preview

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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

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 first 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

impact on the future direction and applications of ambient

ionization.

A working definition 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.

Fig. 1 Illustration of desorption electrospray ionization (DESI).1

a

Department of Chemistry, Purdue University, West Lafayette, Indiana47907, USA. E-mail: [email protected] School of Biomedical Engineering, Purdue University, WestLafayette, Indiana 47907, USAcCenter for Analytical Instrumentation Development, Purdue University,West Lafayette, Indiana 47907, USA

This paper is part of an Analyst themed issue on Ambient MassSpectrometry, with guest editors Xinrong Zhang and Zheng Ouyang.

Demian Ifa

Demian Ifa received his B.S. in

pharmacy from the State

University of Sao Paulo

(UNESP), Brazil. He received

his M.S. in organic chemistryfrom 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

the development of desorption

electrospray ionization and its applications to imaging and quan-

titation.

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 PurdueUniversity-West Lafayette under

the direction of Prof. R. Graham

Cooks. Her research focuses on

practical applications and funda-

mentals 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.

This journal is The Royal Society of Chemistry 2010 Analyst, 2010, 135, 669681 | 669

CRITICAL REVIEW www.rsc.org/analyst | Analyst

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(Note that electrospray ionization (ESI), atmospheric pressure

MALDI, and atmospheric pressure chemical ionization (APCI)

are excluded by this definition.) 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 gov-

erned 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 isaround 2 eV,4 which is similar to that in ESI, so limited frag-

mentation 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 specificity 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 themethod.

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 beionized. 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 likeDESI which depend on a solvent spray, and others like direct

analysis in real time (DART)9 which utilize plasmas to create gas-

phase 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 flowand 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.

Fig. 2 Illustration of low temperature plasma (LTP) probe for desorp-

tion and ionization.13

Zheng Ouyang

Zheng Ouyang received his B.E.

and M.E. degrees in automatic

control from Tsinghua Univer-

sity, 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 Engi-

neering at Purdue University.

His research interests include

the development of miniature

mass spectrometry analysis systems and their applications for

biomedical analysis.

Graham Cooks

Graham Cooks received Ph.D.

degrees from the University of

Natal (now KwaZulu-Natal)

and Cambridge University. His

interests involve instrumenta-

tion, fundamentals and applica-tions of mass spectrometry.

670 | Analyst, 2010, 135, 669681 This journal is The Royal Society of Chemistry 2010

View Article Online
http://dx.doi.org/10.1039/B925257F


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The plasma methods have the advantage relative to the spray

methods of not requiring expendables (a low carrier gas flow,

even air, suffices). 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 isdiscussed 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 solu-

tions too (see Section 6, below) and a case can be made that the

vapor-phase getting of molecules by charged microdroplets15,16

first described by Fenn and Furstenau, represents a vapor-phase

ambient ionization experiment.

2. Key mechanistic features

DESI

The main DESI mechanism has been described as droplet pick-up.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 film colli-

sion event (Fig. 3C).19 Experiments in which DESI spectra are

recorded at 50 Hz might speak to the short time scale of the

fundamental process.20 Note charged droplet spray, which

characterizes DESI, can be produced without application of an

external voltage, a simplified but less efficient procedure.21

3. Solvent, substrate and geometrical effects

Solvent effects

Solvent choice greatly affects the DESI ionization efficiency, anexpected result given the key role of dissolution in the mecha-

nism. 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 efficiently 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 improvingthe 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 surfac-

tants 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 film 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 thenature 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 modification might involve chem-

ical derivatization (reactive DESI, below) or the solvent might be

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 top-

down 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 efficiency.

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.

Geometry effects

There are geometrical requirements for the spray methods

specifically 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 prob-

lematic to some investigators. Two alternatives have been sug-

gested. One involves so-called geometry independent (GI)-

DESI,24 Fig. 4, in which both the incident and emergent spray

angles are fixed and near-normal to the substrate, so no geometry

optimization is needed. This GI-DESI experiment can be

thought of as using reflection geometry. The alternative method

uses transmission mode geometry. In this experiment, the sampleis 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 specificity and sensitivity (compare

reactive DESI, discussed below).

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 significant inefficiencies 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 spec-

trometer were successful in recording high-quality spectra overdistances of 13 m.28 These experiments simply used the mass

spectrometer suction to transport analyte but this is not efficient

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.) Efficiency can be

increased by using a large capacity, low speed supplementary

pump to move large volumes of gas to the MS inlet under

laminar flow conditions, then intercepting the ions. These

conditions allowed 8 m transfer with the lab-scale mass spec-

trometer (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 effi-

ciency 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 sensi-

tivity and high molecular specificity for trace analytes in complex

matrices.

4. Chemical reactions

Reactive DESI

The ambient ionization methods, especially the spray methodslike 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 first used to increase the specificity of ionization of

particular analytes. Examples are given in Fig. 6, and attention is

Fig. 4 Two simplified geometries for DESI. (A) and (B) Reflection 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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drawn to the relatively non-polar analytes like cholesterol,

fat-soluble vitamins (A and D), and anabolic steroids for whichDESI is normally less sensitive. Reactive forms of the plasma-

and 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 formation

3436

and chem-ical bond-forming reactions30,3740 have gained increasing atten-

tion in reactive DESI experiments since they are analogous to

ordinary solution-phase functional group reactions. Other reac-

tion 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. 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

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 significant 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.

Electrochemistry

As is also the case with electrospray ionization, electrochemicalprocesses 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 significantly influ-

ence the DESI mass spectra of organic compounds. One such

case is that of easily oxidized or reduced compounds, e.g. ben-

zoquinone (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 1,4-

hydroquinone and then be ionized to yield the [M H]

even-electron 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 confirmed 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 addi-

tional electrode (the substrate used in DESI, where electro-

chemical redox reactions can occur). It is likely that discharge-

induced electron attachment (see next section) is responsible.

The combination of electrochemistry (EC) and mass spec-

trometry (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 disulfide 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 specific configurations when DESI is per-

formed in air, i.e. discharge can occur between the emitter tip and

inlet capillary of the mass spectrometer, if sufficiently closely

positioned (ca. 1 mm). The occurrence of unintended oxidation

in DESI was first 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 withspray ionization (LTP and DESI). A good example is provided

by the analysis of condensed-phase saturated hydrocarbons

which are difficult 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 dehydro-

genation steps, to generate ketones and alcohols.

Fig. 7 (A) Electrochemical reduction of 1,4-benzoquinone (BQ) under

DESI conditions, as compared to ESSI. (B) Reactive DESI of n-octa-

decane (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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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 measurementswhen 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 ioni-

zation efficiency. 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 flows. By combining DESI and

LTP, oxidation (or other reactions) might be useful in the anal-

ysis 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

endogenous chemicals (inks, drugs of abuse, pharmaceutical

molecules, or biological molecules) in various objects (finger-

prints,52 documents,53 tissue sections,30,5458 etc.). Because of the

ambient nature and softness of DESI ionization, the unmodified

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 withexogenous compounds. DESI tissue imaging to recognize

diseased tissue for tumor diagnostics is an ongoing interest.55 As

shown in Fig. 9, some specific 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 specific solution-phase 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 ante-

rior 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 spectrometryexperiment 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 fluids and tissues make the direct detection and

quantification 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 concen-

trations of hormones in biological fluids (whole blood, plasma,

or serum) could not be achieved. New chemical reactions with

higher reaction efficiency will be established to improve sensi-tivity to allow their detection. Another restriction is that lipids

with relatively low proton affinities or cation affinities could not

be efficiently ionized, and their signals are suppressed by other

lipids. For example, the signal of phosphatidylethanolamines

(PEs), with lower ionization efficiency than phosphocholines

(PCs), is often suppressed by PCs in the positive ion mode of

DESI. Reagents are needed to specifically react with PE to

enhance their signals. On the other hand, reactions can also be

implemented to selectively ionize members of a specific class of

polar lipids in applications where differentiation of compound

classes is needed.

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

flowed 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 disulfide bonds.


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MS/MS imaging

Tandem mass spectrometry provides additional specificity for

many ambient ionization experiments, chemical specificity 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 confirm assignments.

The potential value of MS/MS imaging for this purpose is

apparent from studies of chemical imaging of latent finger-

prints.52 Natural sebaceous oils left as fingerprints can bedetected and identified (Fig. 10A). This observation can be

extended to the identification of illicit drugs, explosives, phar-

maceutical compounds and other chemicals, which have been

handled through the analysis of latent fingerprints, 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 significantly longer to

record for targeted analytes, but they provide greatly increased

chemical specificity.

High mass resolution imaging

An approach to improved molecular imaging that can supple-ment 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 time-

consuming 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 informa-

tion obtained from complex samples.

An example of the value of DESI-MS imaging using

a commercial LTQ/Orbitrap XL mass spectrometer, in dis-

tinguishing isobaric species in the mouse brain (with mass reso-

lution 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

specific 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 spec-

trometry would also increase specificity. 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 experi-

ment increases with the inverse square of the spot size. Onesolution to this, as in the MALDI work of Heeren and

co-workers,63 is to use the microscope rather than the micro-

probe 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 fine needles, which are used to

remove material that can then be examined in the open envi-

ronment66 or an atomically sharp needle can be used to provide

a local site of high field and hence favored desorption. Note that

not all these experiments are amenable to ambient ionization.

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.


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Large area detection

Small area analysis represents one frontier in ambient ionization,

large area analysis another. Large area detection has been

pursued to efficiently 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 ach-

ieved in three ways: (i) rastering the source across the area, which

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 beuseful 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 quantification 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. 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).

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 identified 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.


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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

specificity and the sensitivity of detection. Implementation of

negative ion detection on the miniature mass spectrometer rep-

resented 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 iondetection, it is now possible to achieve using DESI: (a) interro-

gation of larger areas (ca. 6 cm2), (b) increased specificity 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 lowworking pressure of the mass analyzer. Thus, the flow conduc-

tance 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 exper-

iment, DESI was used with a Mini 10 mass spectrometer to detect

25 ng cocaine on a Teflon surface.

6. New methodologies and improved performance

Liquid DESI

Ambient ionization,68 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 solidsamples, 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 exper-

iment 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.

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 biofluid samples such asurine.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 curcumi-

noids were successfully detected in commercially available

functional beverages containing turmeric extract and currypowder by DART-MS.73 The first reports on atrazine determi-

nation in solution were described in the first publication on LTP.

Therapeutic drug analysis from serum

New methods for quantitation of drugs in biological matrices

which reduce the need for sample preparation are being devel-

oped using such direct analysis methods as desorption

Fig. 12 DESI analysis of liquid samples.47

Fig. 13 MS spectra showing the direct DESI-MS analysis of solutions

containing bovine serum albumin (BSA).47 The insets show the corre-

sponding deconvoluted spectra.


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electrospray ionization (DESI).74 The endpoint of these experi-

ments 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 efficacious in most cases, imatinib pharmacoki-

netics shows significant inter-patient variability. Also, there is anestablished relationship between imatinib exposure and the

drugs efficacy 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 concen-

trations. 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 quantification usingminiature 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 pharma-

ceutical industry in storing and transporting blood as spots on

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 associ-

ated 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 incor-porated into the porous medium for controlled elution is being

explored.76,77

Paper spray

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 con-

taining the blood spot (or other biological fluid) 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 fine point). It

is believed that the spray process is very similar to that in an

array of nanospray emitters that the paper serves to filter cells,

that it can have chromatographic and electrophoretic separation

effects, and that the point in the paper speeds up the wick-driven

solvent flow 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 fit. 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, evenmore 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.

Significant 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 applica-

tion, 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.Fig. 15 Calibration curve for imatinib in raw, untreated serum.74

Fig. 14 MS spectrum showing the DESI-MS detection of metham-

phetamine (1 mg/mL) in a raw urine solution.47 DESI spray solvent used

was methanol/acetic acid (1:0.03 by volume).


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Acknowledgements

Support is acknowledged from the National Science Foundation

(NSF 0848650), the Department of Homeland Security (DHS

08116108) and the Office of Naval Research (N00014-05-1-0405)

for support. We also thank Thermo Scientific Instruments, Inc.

and ICx, Inc. for support and valuable interactions. Helpful

comments by Dr Daniel Weston (AstraZeneca R&D Charn-

wood, UK) and Hao Chen (Ohio University) are greatly

appreciated.

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