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7/27/2019 Desorption electrospray ionization and other ambient ionization methods: current progress and preview
1/13
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.
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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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