1. No category

focal point
JOANNA SZPUNAR ,* RYSZARD LOBINSKI, AND ANDREAS PRANGE
CNRS, PAU , FRANCE (J.S., R.L.);
WARSAW UNIVERSITY OF TECHNOLOGY , WARSAW , POLAND (R.L.); AND
GKSS-RESEARCH CENTRE, GEESTHACHT, GERMANY (A.P.)
Hyphenated
Techniques for
Elemental Speciation in
Biological Systems
INT RODUCTIO N
T
he recognition of the fact that
in environmental chemistry,
occupational health, nutrition,
and m edicine the chemical, biological, and toxicological properties of
an element are critically dependent
on the form in which the element occurs in the sample has spurred rapid
development of an area of analytical
chemistry referred to as speciation
analy sis. 1 T he com b ination of a
chrom ato graph ic sep ara tion technique, which ensures that the analyte
compound leaves the column unaccompanied by other species of the
analyte element, with atomic spectrometry, permitting a sensitive and
speciŽ c detection of the target element, has becom e a fundamental
tool for speciation analysis, as discussed in m any review p ublications. 2–7
The classical speciation analysis
discussed in a focal point article in
* Author to whom correspondence should be
sent.
102A
Volume 57, Number 3, 2003
1997 3 has targeted well-deŽ ned analytes, usually anthropogenic organometallic compounds and the products of their environmental degradation, such as methylmercury, alky llead, bu ty l- an d ph en yltin
compounds, and simple organoarsenic and organoselenium species.
C alib ration standards w ere either
available or could be readily synthesized. The presence of a metal(loid)–
carbon covalent bond assured a reasonable stability of the analyte(s)
during sample preparation. The volatility of the species allowed the use
of gas chromatography with its inherent advantages, such as the high
separation efŽ ciency and the absence
of the condensed m obile phase that
enabled a sensitive (down to femtogram levels) element-speciŽ c detection by atomic spectroscopy. 8,9
A to tally differen t situation is
faced by the analyst interested in endogenous metal species in biological
systems.6,10 –13 M illions of years of
evolution have resulted in a great variety of biological ligands with a signiŽ cant coordinating potential for
trace elements. They include small
organic ligands (e.g., citrate, tartrate,
oxalate, or phytate, aminoacids, and
oligopeptides), m acrocyclic chelating molecules, and macromolecules,
such as proteins, DNA restriction
fragments, or polysaccharides. The
complexity and the usually poor understanding of the system (the majority of trace element species with
biological ligands have not yet been
discovered!) often m akes even the
deŽ nition of the target analyte problematic. The generally poor volatility
of the m etal coordination complexes
with biological ligands by comparison with organometallic species calls
for separation techniques with a condensed mobile phase that negatively
affects the separation efŽ ciency and
the detection limits.
R ecent im pressive prog re ss toward lower detection limits in inductively coupled plasma m ass spectrometry (IC P-M S), toward higher
resolution in separation techniques,
especially capillary electrophoresis
and electrochromatography, and toward higher sensitivity in electros-
FIG. 1. Evolution of the concept of speciation analysis: from the determination of anthropogenic organometallic contaminants toward the molecular description of trace element reaction mechanisms. The information contained in the genome (the set of genes
carried by an organism) is expressed in the proteome (all the proteins produced from all the genes of a genome). Enzymatic proteins activated by a metal are responsible for the synthesis of metabolites (metal-binding ligands), and the entire set of metabolites
is referred to as metabolome. The elucidation of a reaction mechanism includes the identiŽcation of the metal-binding metabolite,
the enzyme, and the coding gene.
pray mass spectrometry for molecule-speciŽ c detection at trace levels
in co m plex m atrices allo w s n ew
frontiers to be crossed. This applies
in particular to the identiŽ cation and/
or structural characterization of endogenous species of essential, beneŽ cial, and toxic elements and to metabolism studies of m etal probes in
biology and m edicine. Indeed, the
extreme complexity of the matrix,
the trace concentrations present, and
the non-availability of calibra tion
standards have been powerful limiting factors in the acquisition of speciation-relevant inform ation in these
areas.
As shown schematically in Fig. 1,
th e fo cus o f speciation-relevant
research is shifting from the determ in ation of anth ro pog enic m etal
species and the products of their en-
vironmental degradation to endogenous and biosynthesized m etal species. The ultimate objective is an understanding of the mechanisms controlling the essentiality and toxicity
of trace elements in biological systems at the m olecular level. This can
be achiev ed b y com pleting the
chemical information obtained by
the identiŽ cation of genes directly
triggering the biosynthesis of metalbinding ligands (e.g., m etallothioneins), or coding for enzymes activated by metals to produce as a metabolite a metal binding ligand (e.g.,
phytochelatins). This focal point article describes the relevant analytical
challenges, discusses the state-ofthe-art of suitable analytical techniques, and highlights the trends that
are aimed at the integration of m o-
lecular biology approaches into analytical spectrometry.
TRACE ELEM ENT SPECIES IN
BIOLOGICAL SYSTEM S:
ANALYTICAL TARGETS
Figure 2 overviews trace elements
with an identiŽ ed role in biological
systems. Some metals have the notoriety of showing either acute (e.g.,
Hg) or chronic (e.g., Pb) toxicity,
whereas others (e.g., Mo, Mn, Fe,
Co, Cu, Zn), referred to as essential,
are needed for the accomplishment
of life processes. 14 Some elements
(e.g., V, Cr, Ni) are recognized as
beneŽ cial to life, although the borderline between being essential or
beneŽ cial is vague. A number of elements show a dual character: they
are essential in one oxidation state,
APPLIED SPECTROSCOPY
103A
focal point
FIG. 2. Trace elements of interest in medicine and biology: endogenous trace element species in biological systems.
e.g., Cr(III) or Se(IV), and yet toxic
in the other state, e.g., Cr(VI) or
Se(VI). Some elements, e.g., Co, can
be considered essential only if present in a particular organic form, e.g.,
as cyan ocobalam in (v itam in B 1 2 ),
and are toxic when in other form s.
On the other hand, As, a notoriously
to xic elem ent, b eco m es harm less
when present in the form of, e.g., arsenobetaine. A separate class consists of the m etals used as pharm acological probes. Platinum (cisplatin , carbo platin) an d R u 3 1 ( f a cRuCl 3(NH 3 ) 3) compounds are used in
cancer therapy, whereas some Au
com pounds (a urithiom alate, auroth iog luco se) are im po rtant an tiarthritic drugs.15 A wide range of Tc
compounds (e.g., Tc-labeled antibodies, Tc-mercaptoacetyl glycine complex) are used for diagnostic imaging
of renal, cardiac, and cerebral functions and various form s of cancer.
The occurrence of a free metal
ion, especially of a transition element, in a biological cell rich in ligands with a signiŽ cant coordinating
potential is highly improbable. M et104A
Volume 57, Number 3, 2003
alloids (As, Se) are known to be metabolized by living organisms in a
way that leads to the formation of a
covalent bond between the heteroatom and the carbon incorporated in
large structures (e.g., arsenosugars,
selenoproteins). Metals are usually
present in the form of coordination
complexes, of which some, e.g., cyanocobalamine, are remarkably stable. M etal com plexation by proteins
via nitrogen or oxygen confers the
activity to several enzymes, whereas
the coordination via a sulfur atom is
usually associated with the detoxiŽ cation of heavy m etals. M etals that
activate an enzyme, e.g., nicotianamine synthase (Fe, Ni) or phytochelatin synthase (C d), are usu ally
found to be complexed by the metabolite nicotianamine or phytochelatin, respectively. The quantity of
the synthesized m etabolite regulates
th e co ncentration o f th e elem ent
available for the enzymatic reaction.
Relatively little is known about
the relevance of m etal coordination
to lipids and carbohydrates, although
the potentially negatively charged
oxygen functions and polyhydroxy
groups can bind cations electrostatically and by chelation, respectively.
The complexation of divalent cations
by the carboxylic acid groups of
uronic acids from plant cell walls is
well established. The analytical challenges faced in the area of metal
probes include both the identiŽ cation
of the products of m etallodrug metabolism and the understanding of
the binding of metallodrugs to transport proteins and DNA fragments.16
ANALYTICAL CHALLENGES
AND ANSW ERS
The low concentration of the trace
element present in a biological tissue
(usually below 1 mg g 2 1 ) and the
complexity of the m atrix (a chromatographic fraction may still contain a larger number of compounds
present above the 1 nM level in the
original sample) represent the two
major challenges to element speciation analysis in biological systems.
The Ž rst problem concerns the selectivity of the separation technique
FIG. 3. Hyphenated techniques in trace element speciation analysis in biological systems.
allowing the target analyte species to
arrive at the detector well separated
from other species of the element of
interest. Indeed, two metalloproteins
that only differ by one amino acid
can have different m etal-complexing
properties, and hence, play different
biochemical roles. The second problem involves the detection sensitivity. The already low concentrations of
many endogenous trace elements in
biological systems are usually distributed among several species in
which the contribution of the m etal(loid) to the total structure is m inute in terms of weight.
The above challenges can be addressed by an analytical strategy
based on a properly chosen hyphen-
ated technique of which the choices
available are schematically shown in
Fig. 3. In the simplest case a separation technique such as chromatography, electrochromatography, or gel
electrop horesis is com b ined w ith
ICP-MS. The coupling is realized
via a nebulizer (for column techniques) or by laser ablation (for planar techniques).
The separation component of the
coupled system becomes of particular concern when the targeted species have closely similar physicochemical properties. Column techniques, hig h-perfo rm an ce liquid
chromatography (HPLC) and capillary electrophoresis (CE), are the
usual choice because of the ease of
on-line coupling and the variety of
separation m echanisms and m obile
phases available allowing the preservation of the species identity. The
denaturating conditions of sodium
do decysu lfate polyacr ylam id g el
electrophoresis (SDS PAGE) usually
prevent its application to metal coordination complexes with proteins,
but two-dimensional gel electrophoresis is indispensable in seleno- and
phosphoroproteomics because of its
impressive peak capacity.
For element-speciŽ c detection, inductively coupled plasma mass spectrometry is virtually the only technique capable of coping, in on-line
mode, with the trace element concentrations in biological materials.
APPLIED SPECTROSCOPY
105A
focal point
The fem togram level absolute detection limits m ay turn out to be insufŽ cient if an element present at the
ng/mL level is split into a number of
species, or when the actual sample
amount analyzed is limited to several
nanoliters, as in the case of CE. The
isotope speciŽ city of ICP-M S offers
a still under-exploited potential for
tracer studies and for improved accuracy in quantiŽ cation via the use
of isotope dilution techniques.
The third important component of
the analytical strategy is the identiŽ cation and characterization of metallosp ecies, either new ly discov ered, or those that may have been reported but for which standards are
unavailable. M olecule-speciŽ c detection can be achieved by electrospray MS or matrix assisted laser deso rp tio n io nization tim e-of- igh t
(MALDI-TOF) MS for column or
planar separation techniques, respectively. Structural information can be
acquired by collision-induced dissociation (CID) of an ion selected by a
quadrupole mass Ž lter followed by a
product ion scan using a quadrupole,
ion-trap, or a TOF m ass analyzer.
In d u ctively C o u p led Plasm a
M ass Spectrometry Detection in
C h ro m ato grap h y an d C ap illary
Electrophoresis. The separation of
picogram or nanogram quantities of
m etallo-com po und s has n ot yet
reached m aturity, and m any phenomena, such as metal adsorption or
ligand exchange, at these levels are
still poorly understood. The principal
HPLC separation mechanisms used
in bioinorganic speciation analysis
include size-exclusion, ion-exchange,
and reversed-phase chromatography.
Capillary electrophoresis is less mature but offers exciting possibilities
for speciation analysis owing to its
high separation efŽ ciency, the nanoliter sample requirement, and the absence of packing susceptible to interact with metals and to affect the
complexation equilibria.17,18 The combination of electrophoretic and electroosmotic  ows provides the ability
to separate a wide variety of positive, neutral, and negative ions and
compounds in one run.
The use of a quadrupole mass an106A
Volume 57, Number 3, 2003
alyzer in ICP-MS detection is the
most widespread. The latest generation of instruments offers sub-fem togram absolute detection levels for
many m etals. The isobaric overlaps
are generally not a problem because
of the on-line separation from the
po tential in terferents, e.g., C l
( 40Ar 35Cl) in the case of 75 As determination, but ghost peaks may appear. The application of a double-focusing sector-Ž eld instrument offers
the higher resolution that may be required for the interference-free determination of sulfur or of the isotope ratios of some elements, e.g.,
Cr, Fe, and V.19 An increase in resolution inevitably leads, however, to
a dramatic decrease in sensitivity. It
should also be noted that the sensitivity of the latest generation quadrupole instruments is only a factor of
2–3 lower than that of high-resolution ICP-M S operated in the low resolution mode. A good tradeoff between sensitivity, freedom from isobaric interferences, and price is offere d by IC P -M S instrum ents
equipped with a collision cell that
have recently proliferated on the
market. 20
Both quadrupole and sector-Ž eld
mass spectrometers are scanning (sequential) analyzers and multi-isotope
analysis can be achieved at the expense of the measurement sensitivity
and precision. The sequential measurement of m /z values at different
points within a time-dependent concentration proŽ le of a transient signal can result in peak distortions and
quantiŽ cation errors, comm only referred to as spectral skew. 21 The alternative is TOF-MS, which features
the ability to produce a complete
atomic mass spectrum in less than 50
ms and thus allows the recording of
very brief transient signals with high
Ž delity. 21 This is especially useful in
the on-line isotope ratio determination, but a 10-fold loss in sensitivity
of an ICP-TOF-M S instrument in
comparison with the latest quadrupole instruments m ay create an obstacle for the wider application of
ICP-TOF-MS as a detector in the
capillary electrophoresis of metallobiomolecules in biological systems.
The key to a succesful HPLC/CE–
ICP-MS coupling is the interface.
Figure 4 shows schematically the
most frequently used combinations.
In the simplest case, the exit of an
HPLC colum n (i.d. 4.6–10 mm) is
connected to a conventional pneumatic or cross ow nebulizer. The use
of capillary or m egabore (0.32–1.0
mm ) HPLC systems, which are becoming popular especially for reversed-phase chrom atogra phy, requires the use of m icronebulizers, either direct injection (DIN, DIHEN)
or ones (e.g., M icrom ist) Ž tted with
a small-volume nebulization chamber. The CE–ICP-M S coupling is
less straightforward. The problems
due to the laminar  ow generated by
the nebulizer suction, loss of sensitivity because of the electroosm otic
 ow dilution by the m akeup liquid,
and peak broadening in the spray
chamber have been resolved in the
com m ercially av ailable inter fa ce
based on a total-consumption selfaspirating m icronebulizer Ž tted with
a small-volume spray chamber. 22,23
Figures 5–7 show three representative examples of the use of HPLC/
CE–ICP-M S in the speciation analysis of metallobiomolecules. Sizeexclusion chrom atography coupled
to ICP-M S allows the m onitoring of
the presence of stable metal complexes in liquid samples, e.g., tissue
cytosols. 24 The peak width is sufŽ ciently large to allow the use of a
quadrupole mass analyzer for the simultaneous m onitoring of up to 12
isotopes. The correlation of the elution vo lum e w ith the m olecu lar
weight of the eluted molecule allows
the determination of the m olecular
weight of the analyte. On this basis,
a hypothesis regarding the identity of
the eluted species can be put forward. Figure 5 presents a case in
which the identity of a Pb–di-rhamnogalacturonane complex in wine is
conŽ rmed by the observed co-elution of elements (Ba, Ce, Sr) with a
characteristic ionic radius Ž tting the
cavity of the rhamnogalacturonane
dimer. 25
A Ž ner characterization of mixtures of hydrophobic metal com plexes or of metal-containing protein
FIG. 4. Instrumental setups for HPLC and capillary electrophoresis with ICP-MS detection.
fractions isolated by size-exclusion
chromatography can be achieved by
capillar y/m egabore reversed-phase
HPLC. 26 Very sharp peaks can be
obtained, as illustrated in Fig. 6,
which shows the determination of
cyanocobalamin and its analogues
by HPLC–ICP-MS interfaced via a
direct injection nebulizer. The third
example (Fig. 7) shows a mass  ow
CE–ICP-M S electropherogram of a
rabbit liver preparation of metallothionein. 27 The individual isoforms
are separated by capillary electrophoresis, whereas S, Cd, and Zn are
quantiŽ ed on-line by isotope-dilution
ICP-MS. In this way, the stoichiometry of the m etal–protein complex
can be determined. 27,28
E lectrosp ray -Ion iza tion M a ss
Spectrometry/M ass Spectrometry
Detection in Chromatography and
C a p illary E lectr o ph o resis. T he
identiŽ cation of unknown species
detected by ICP-M S can be achieved
by running, in parallel, the same separation using electrospray MS/MS
detection. In the standard instrumental setup (Fig. 8), a low- ow sepa-
ration technique, capillary HPLC or
CE, is used. Another run is often carried out with a post-column acidiŽ cation that allows the cleaving of the
metal off the ligand and thus enables
the direct determination of the m olecular weight of the latter. 29 When a
calibration standard of the analyte is
available, a simple quadrupole mass
analyzer is usually sufŽ cient for the
conŽ rmation of the analyte identity.
A Ž ner characterization of unknown
bioligands requires a tandem MS
(e.g., triple quad or Q-TOF) or an
MS n (e.g., ion-trap MS) instrument.
Figure 9 shows an example of the
id en tiŽ cation of a m etal-b in din g
polypeptide and its complexes with
Cd 21 by reversed-phase HPLC–ESIMS. In ESI-M S, proteins (and protein–metal complexes) show a characteristic envelope of peaks due to
multiple ionization that allows the
accurate (60.5 Da) determination of
the m olecular weight (Fig. 9a). The
mass spectrum taken at the apex of
a peak during chromatography of
rabbit liver metallothionein preparation (Fig. 9b) allows the determina-
tion of the molecular weight of the
metal–metallothionein complex.30 Another m ass spectrum (Fig. 9c), obtained after on-line acidiŽ cation of
the chromatographic ef uent, allows
the determination of the m olecular
mass of the apo- (metal-free) metallothionein and thus allows a search
for its identity in a database. 29 The
difference between the M r of the
complex and M r of the ligand is a
measure of the complex stoichiometry and can be used for the validation of data obtained in the experiment outlined in Fig. 7.
Another example (Fig. 10) shows
a Ž ne characterization by CE–ESIMS/M S of a Cd containing fraction
(isolated by size-exclusion chromatography (SEC)) of the cytosol of
soybean cells exposed to a stress of
Cd 21 . 31 The total ion-current electropherogram shows a number of peaks
to which a m olecular m ass value can
be attributed. The identiŽ cation of
the compounds, in this case a series
of polypeptides referred to as phytochelatins, is possible by acquiring
on-line M S/M S data. The interpreAPPLIED SPECTROSCOPY
107A
focal point
FIG. 5. Speciation analysis of trace elements in wine by size-exclusion chromatography with multielement detection by ICP-MS.25 The arrows indicate elution of the molecular weight markers (pollulans) used to calibrate the column. The hydrodynamic volume and the coelution of Ba, Ce, Pb, and Sr suggest the elution of a metal complex
with the dimer of rhamnogalacturonan (partly shown in the inset).
tation of the CID mass spectrum of
the protonated molecular ion responsible for a peak in the CE–ESI electropherogram allows the determination of the amino acid sequence of
each of the separated polypeptides.
M ultidim en sional H yph en ate d
Techniques in Speciation Analysis.
The complexity of the biological matrix m ay require the combination of
two or m ore separation mechanisms
in series to assure that a unique m etal species arrives at the detector at a
given time. This approach is illustrated in Fig. 11, which shows an example of the identiŽ cation of an arsenic species in a marine biota. The
individual arsenic compounds are
isolated by tri-dimensional LC includ in g size-exclu sio n, an ion -ex change, and cation-exchange mechanisms. The isolated species are analyzed by electrospray TOF-M S; the
accuracy of the m olecular m ass measurement (especially important in the
case of compounds of As, which is
a monoisotopic element) and the
matching of the isotopic pattern of
the molecular ion allow the identiŽ cation of the compound. The identity
conŽ rmation or identiŽ cation of an
unknown com pound can be achieved
by CID-M S.
M OLECULAR DESCRIPTION
OF M ECHANISM S OF TRACE
ELEM ENT INVOLVEM ENT IN
BIOLOGICAL SYSTEM S
FIG. 6. Reversed-phase HPLC–ICP-MS chromatogram of cyanocobalamin and its analogues obtained using a microbore C 8 column coupled to ICP-MS via a direct injection
nebulizer.32
108A
Volume 57, Number 3, 2003
Hyphenated techniques allow the
identiŽ cation in a biological sample
of a metal-complexing metabolite
coded directly by a gene (e.g., metallothioneins), or biosynthesized by
a metal-activated enzyme coded by a
gen e (e.g., phy toch elatin s, citric
acid, nicotianamine) (see, for instance, Fig. 1). In this context, the
role of molecular biology techniques
for the isolation and cloning of the
corresponding gene is rapidly growing in importance. The identiŽ cation
of the metabolite (m etal-binding ligand) as functionally important, e.g.,
in a hyperaccumulating plant, should
be accompanied by the identiŽ cation
of the relevant gene, its cloning, and
its expression in a simple organism,
such as bacteria or yeast. The result
FIG. 7. Mass ow multielement CE–ICP-MS electropherograms of a rabbit liver metallothionein preparation obtained using the CETAC CEI-100 interface and quantiŽcation
by isotope dilution.27 The quantities given refer to the amount of protein producing the
corresponding peak. The spatial structure of the molecule, eluting as the peak highlighted in yellow, is shown in the inset of the Žgure.
of an identiŽ cation achieved by a hyphenated technique may therefore
only be considered validated if the
gen etically m o diŽ ed b acteria or
yeast exposed to metal stress will
biosynthesize the same metallocompound as the original plant or animal.
Note also that the isolation, cloning, and expression of functional
genes may be considered as an advanced sample preparation m ethod
for speciation analysis. It allows the
exchange of the complex matrix of a
plant or animal cytosol into a simpler
matrix of bacteria or yeast extract
with the simultaneous enrichm ent of
the m etallospecies of interest. In this
way, a hyphenated technique will be
used to conŽ rm the presence of an
expected species rather than to identify it among thousands of other bioligands.
CONCLUSION
The decreasing detection limits of
ICP-MS, the availability of efŽ cient
interfaces to HPLC and capillary
FIG. 8. Instrumental setups for HPLC and CE with electrospray MS and MS/MS detection.
APPLIED SPECTROSCOPY
109A
focal point
FIG. 9. Determination of the molecular mass and the degree of metallation of metallothioneins by reversed-phase HPLC with electrospray MS detection.29 The inset (a) gives
the principle of the molecular mass determination of a protein and of its complex with
a metal. Insets (b) and (c) give mass spectra obtained at the apex of peak 4 of the
chromatogram in a setup without and with post-column acidiŽcation, respectively.
electrophoresis, and the increasing
sensitivity of electrospray M S for
molecule-speciŽ c detection at trace
levels open the way to the characterization of endogenous trace element
species in biological systems and offer the key to the understanding of
many chemical processes essential
for life. The development of analytical m ethods for biochemical speciation analysis is being carried out at
the crossroads of interest of m any
disciplines and can proŽ t from the
interdisciplinarity of approach to the
same degree that it can suffer from
the lack of it. In order to cope with
the complexity of biological matrices, m ultidimensional separation and
detection approaches are required.
The elucidation of the role of a metal
sp ecies iden tiŽ ed by m ultidim ensional analytical techniques in a biological system requires the identiŽ cation of the gene coding the correspo nding b ioligand d irectly, or
coding an enzyme responsible for its
synthesis, the isolation of the gene,
and its expression in a bacteria or
yeast. A positive identiŽ cation of the
metabolite, previously identiŽ ed in
the original plant or animal tissue, in
the m utant bacteria or the yeast, will
deliver the ultimate proof of its involvement in the metal metabolism.
The more powerful analytical techniques make the question about the
preservation of the original form
more pertinent than ever; direct speciation analysis of liquid sampled
from an individual cell appears as a
long-term goal.
ABBREVIATIONS
AES
TOF
CE
CEC
CID
FIG. 10. CE–ESI-MS/MS characterization of polypeptides in a Cd containing fraction
isolated from the cytosol of soybean cells by size-exclusion chromatography. 31 A total
ion current CE–ESI-MS electropherogram is shown. The mass spectrum at the apex of
a peak allows the determination of the molecular weight of the peptide present. The
electropherogram acquired using CID of the protonated ions of the molecules found by
CE–ESI allows the determination of the amino acid sequence of the peptides detected.
An example structure of the major compound found (/b-alanine-PC 3 ) is shown.
110A
Volume 57, Number 3, 2003
DIN
DIHEN
ESI
FT-IC R
atom ic em ission spectrometry
time-of- ight
capillary electrophoresis
capillary electrochromatography
collision-induced dissociation
direct-injection nebulizer
direct-injection high-efŽ ciency nebulizer
electrospray ionization
F ou rier transform ion
cyclotron resonance
FIG. 11. IdentiŽcation of an arsenosugar by multidimensional hyphenated techniques. (a) PuriŽcation of the arsenic compound: the
As-containing fraction from SEC was puriŽed by anion-exchange chromatography. Each of the anion-exchange chromatographic
fractions was further puriŽed by cation-exchange HPLC. Arsenospecies eluted from the cation-exchange column were analyzed by
ESI-TOF-MS; (b) electrospray TOF mass spectra: the vicinity of a peak corresponding to an arsenic-containing compound was
zoomed to reveal the isotopic pattern. The error of the Mr determination represents the difference between the calculated and measured molecular mass values; (c) CID-TOF mass spectrum of the protonated ion of the organoarsenic compound isolated by a quadrupole Žlter. The structure correponds to an arsenosugar: 3-[5 9-deoxy-5 9-(dimethylarsinoyl-b-ribofuranosyloxy]-2-hydroxypropylene
glycol.
APPLIED SPECTROSCOPY
111A
focal point
HPLC
ID
ICP
MC
MEKC
MT
PAGE
Q
q
SDS
SF
high-performance liquid
chromatography
isotope dilution
in ductively
co up led
plasma
multicollector
m icellar electrokinetic
chromatography
metallothionein
polyacrylamide gel electrophoresis
qu adrupo le used as a
mass analyzer
qu adrupo le used as a
collision cell
so diu m d odecyl sulfonate
sector Ž eld
6.
7.
8.
9.
10.
11.
12.
13.
14.
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G. Danielsson, H. Muntau, H. P. van
Leeuven, and R. Lobinski, Pure Appl.
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2. M . J. Tomlinson, L. Lin, and J. A. Caruso,
Analyst (Cam bridge, U.K.) 120, 583
(1995).
3. R. Lobinski, Appl. Spectrosc. 51, 260A
(1997).
4. G. K. Zoorob, J. W. McKiernan, and J. A.
Caruso, M ikrochim. Acta 128, 145
(1998).
5. J. A. Caruso, K. L. Sutton, and K. L. Ack-
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Volume 57, Number 3, 2003
15.
16.
17.
18.
ley, in Comprehensive Analytical Chemistry, D. Barcelo , Ed. (Elsevier, Amsterdam, 2000).
J. Szpunar, Analyst (Cambridge, U.K.)
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