Hyphenated Techniques for Elemental Speciation in Biological Systems

102A Volume 57, Number 3, 2003

focal pointJOANNA 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.)

HyphenatedTechniques for

Elemental Speciation inBiological Systems

INTRODUCTION

T he recognition of the fact thatin environmental chemistry,occupational health, nutrition,

and medicine the chemical, biologi-cal, and toxicological properties ofan element are critically dependenton the form in which the element oc-curs in the sample has spurred rapiddevelopment of an area of analyticalchemistry referred to as speciationanalysis .1 The combination of achromatographic separation tech-nique, which ensures that the analytecompound leaves the column unac-companied by other species of theanalyte element, with atomic spec-trometry, permitting a sensitive andspeci� c detection of the target ele-ment, has become a fundamentaltool for speciation analysis, as dis-cussed in many review publica-tions.2–7

The classical speciation analysisdiscussed in a focal point article in

* Author to whom correspondence should besent.

19973 has targeted well-de� ned an-alytes, usually anthropogenic organ-ometallic compounds and the prod-ucts of their environmental degra-dation, such as methylmercury, al-kyllead, buty l- and phenyltincompounds, and simple organoar-senic and organoselenium species.Calibration standards were eitheravailable or could be readily synthe-sized. The presence of a metal(loid)–carbon covalent bond assured a rea-sonable stability of the analyte(s)during sample preparation. The vol-atility of the species allowed the useof gas chromatography with its in-herent advantages, such as the highseparation ef� ciency and the absenceof the condensed mobile phase thatenabled a sensitive (down to femto-gram levels) element-speci� c detec-tion by atomic spectroscopy.8,9

A totally differen t situation isfaced by the analyst interested in en-dogenous metal species in biologicalsystems.6,10–13 Millions of years ofevolution have resulted in a great va-riety of biological ligands with a sig-ni� cant coordinating potential for

trace elements. They include smallorganic ligands (e.g., citrate, tartrate,oxalate, or phytate, aminoacids, andoligopeptides), macrocyclic chelat-ing molecules, and macromolecules,such as proteins, DNA restrictionfragments, or polysaccharides. Thecomplexity and the usually poor un-derstanding of the system (the ma-jority of trace element species withbiological ligands have not yet beendiscovered!) often makes even thede� nition of the target analyte prob-lematic. The generally poor volatilityof the metal coordination complexeswith biological ligands by compari-son with organometallic species callsfor separation techniques with a con-densed mobile phase that negativelyaffects the separation ef� ciency andthe detection limits.

Recent impressive progress to-ward lower detection limits in induc-tively coupled plasma mass spec-trometry (ICP-MS), toward higherresolution in separation techniques,especially capillary electrophoresisand electrochromatography, and to-ward higher sensitivity in electros-

APPLIED SPECTROSCOPY 103A

FIG. 1. Evolution of the concept of speciation analysis: from the determination of anthropogenic organometallic contaminants to-ward the molecular description of trace element reaction mechanisms. The information contained in the genome (the set of genescarried by an organism) is expressed in the proteome (all the proteins produced from all the genes of a genome). Enzymatic pro-teins activated by a metal are responsible for the synthesis of metabolites (metal-binding ligands), and the entire set of metabolitesis referred to as metabolome. The elucidation of a reaction mechanism includes the identi�cation of the metal-binding metabolite,the enzyme, and the coding gene.

pray mass spectrometry for mole-cule-speci� c detection at trace levelsin complex matrices allows newfrontiers to be crossed. This appliesin particular to the identi� cation and/or structural characterization of en-dogenous species of essential, bene-� cial, and toxic elements and to me-tabolism studies of metal probes inbiology and medicine. Indeed, theextreme complexity of the matrix,the trace concentrations present, andthe non-availability of calibrationstandards have been powerful limit-ing factors in the acquisition of spe-ciation-relevant information in theseareas.

As shown schematically in Fig. 1,the focus of speciation-relevantresearch is shifting from the deter-mination of anthropogenic metalspecies and the products of their en-

vironmental degradation to endoge-nous and biosynthesized metal spe-cies. The ultimate objective is an un-derstanding of the mechanisms con-trolling the essentiality and toxicityof trace elements in biological sys-tems at the molecular level. This canbe achieved by completing thechemical information obtained bythe identi� cation of genes directlytriggering the biosynthesis of metal-binding ligands (e.g., metallothio-neins), or coding for enzymes acti-vated by metals to produce as a me-tabolite a metal binding ligand (e.g.,phytochelatins). This focal point ar-ticle describes the relevant analyticalchallenges, discusses the state-of-the-art of suitable analytical tech-niques, and highlights the trends thatare aimed at the integration of mo-

lecular biology approaches into an-alytical spectrometry.

TRACE ELEMENT SPECIES INBIOLOGICAL SYSTEMS:ANALYTICAL TARGETS

Figure 2 overviews trace elementswith an identi� ed role in biologicalsystems. Some metals have the no-toriety 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 accomplishmentof life processes.14 Some elements(e.g., V, Cr, Ni) are recognized asbene� cial to life, although the bor-derline between being essential orbene� cial is vague. A number of el-ements show a dual character: theyare essential in one oxidation state,


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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 toxicin the other state, e.g., Cr(VI) orSe(VI). Some elements, e.g., Co, canbe considered essential only if pres-ent in a particular organic form, e.g.,as cyanocobalamin (v itamin B 12),and are toxic when in other forms.On the other hand, As, a notoriouslytoxic element, becomes harmlesswhen present in the form of, e.g., ar-senobetaine. A separate class con-sists of the metals used as pharma-cological probes. Platinum (cisplat-in , carboplatin) and Ru31 ( f ac-RuCl3(NH3)3) compounds are used incancer therapy, whereas some Aucompounds (aurithiomalate, auro-th ioglucose) are important an tiar-thritic drugs.15 A wide range of Tccompounds (e.g., Tc-labeled antibod-ies, Tc-mercaptoacetyl glycine com-plex) are used for diagnostic imagingof renal, cardiac, and cerebral func-tions and various forms of cancer.

The occurrence of a free metalion, especially of a transition ele-ment, in a biological cell rich in li-gands with a signi� cant coordinatingpotential is highly improbable. Met-

alloids (As, Se) are known to be me-tabolized by living organisms in away that leads to the formation of acovalent bond between the hetero-atom and the carbon incorporated inlarge structures (e.g., arsenosugars,selenoproteins). Metals are usuallypresent in the form of coordinationcomplexes, of which some, e.g., cy-anocobalamine, are remarkably sta-ble. Metal complexation by proteinsvia nitrogen or oxygen confers theactivity to several enzymes, whereasthe coordination via a sulfur atom isusually associated with the detoxi� -cation of heavy metals. Metals thatactivate an enzyme, e.g., nicotiana-mine synthase (Fe, Ni) or phytoche-latin synthase (Cd), are usuallyfound to be complexed by the me-tabolite nicotianamine or phytoche-latin, respectively. The quantity ofthe synthesized metabolite regulatesthe concentration of the elementavailable for the enzymatic reaction.

Relatively little is known aboutthe relevance of metal coordinationto lipids and carbohydrates, althoughthe potentially negatively charged

oxygen functions and polyhydroxygroups can bind cations electrostati-cally and by chelation, respectively.The complexation of divalent cationsby the carboxylic acid groups ofuronic acids from plant cell walls iswell established. The analytical chal-lenges faced in the area of metalprobes include both the identi� cationof the products of metallodrug me-tabolism and the understanding ofthe binding of metallodrugs to trans-port proteins and DNA fragments.16

ANALYTICAL CHALLENGESAND ANSWERS

The low concentration of the traceelement present in a biological tissue(usually below 1 mg g21) and thecomplexity of the matrix (a chro-matographic fraction may still con-tain a larger number of compoundspresent above the 1 nM level in theoriginal sample) represent the twomajor challenges to element specia-tion analysis in biological systems.

The � rst problem concerns the se-lectivity of the separation technique


FIG. 3. Hyphenated techniques in trace element speciation analysis in biological systems.

allowing the target analyte species toarrive at the detector well separatedfrom other species of the element ofinterest. Indeed, two metalloproteinsthat only differ by one amino acidcan have different metal-complexingproperties, and hence, play differentbiochemical roles. The second prob-lem involves the detection sensitivi-ty. The already low concentrations ofmany endogenous trace elements inbiological systems are usually dis-tributed among several species inwhich the contribution of the met-al(loid) to the total structure is mi-nute in terms of weight.

The above challenges can be ad-dressed by an analytical strategybased on a properly chosen hyphen-

ated technique of which the choicesavailable are schematically shown inFig. 3. In the simplest case a sepa-ration technique such as chromatog-raphy, electrochromatography, or gelelectrophoresis is combined withICP-MS. The coupling is realizedvia a nebulizer (for column tech-niques) or by laser ablation (for pla-nar techniques).

The separation component of thecoupled system becomes of particu-lar concern when the targeted spe-cies have closely similar physico-chemical properties. Column tech-niques, high-performance liquidchromatography (HPLC) and capil-lary electrophoresis (CE), are theusual choice because of the ease of

on-line coupling and the variety ofseparation mechanisms and mobilephases available allowing the pres-ervation of the species identity. Thedenaturating conditions of sodiumdodecysulfate polyacrylamid gelelectrophoresis (SDS PAGE) usuallyprevent its application to metal co-ordination complexes with proteins,but two-dimensional gel electropho-resis is indispensable in seleno- andphosphoroproteomics because of itsimpressive peak capacity.

For element-speci� c detection, in-ductively coupled plasma mass spec-trometry is virtually the only tech-nique capable of coping, in on-linemode, with the trace element con-centrations in biological materials.


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The femtogram level absolute detec-tion limits may turn out to be insuf-� cient if an element present at theng/mL level is split into a number ofspecies, or when the actual sampleamount analyzed is limited to severalnanoliters, as in the case of CE. Theisotope speci� city of ICP-MS offersa still under-exploited potential fortracer studies and for improved ac-curacy in quanti� cation via the useof isotope dilution techniques.

The third important component ofthe analytical strategy is the identi-� cation and characterization of me-tallospecies, either newly discov-ered, or those that may have been re-ported but for which standards areunavailable. Molecule-speci� c de-tection can be achieved by electro-spray MS or matrix assisted laser de-sorp tion ionization time-of-� ight(MALDI-TOF) MS for column orplanar separation techniques, respec-tively. Structural information can beacquired by collision-induced disso-ciation (CID) of an ion selected by aquadrupole mass � lter followed by aproduct ion scan using a quadrupole,ion-trap, or a TOF mass analyzer.

Inductively Coupled Plasm aMass Spectrometry Detection inChromatography and CapillaryElectrophoresis. The separation ofpicogram or nanogram quantities ofmetallo-compounds has not yetreached maturity, and many phe-nomena, such as metal adsorption orligand exchange, at these levels arestill poorly understood. The principalHPLC separation mechanisms usedin bioinorganic speciation analysisinclude size-exclusion, ion-exchange,and reversed-phase chromatography.Capillary electrophoresis is less ma-ture but offers exciting possibilitiesfor speciation analysis owing to itshigh separation ef� ciency, the nano-liter sample requirement, and the ab-sence of packing susceptible to inter-act with metals and to affect thecomplexation equilibria.17,18 The com-bination of electrophoretic and elec-troosmotic � ows provides the abilityto separate a wide variety of posi-tive, neutral, and negative ions andcompounds in one run.

The use of a quadrupole mass an-

alyzer in ICP-MS detection is themost widespread. The latest genera-tion of instruments offers sub-fem-togram absolute detection levels formany metals. The isobaric overlapsare generally not a problem becauseof the on-line separation from thepotential in terferents , e.g., C l(40Ar35Cl) in the case of 75As deter-mination, but ghost peaks may ap-pear. The application of a double-fo-cusing sector-� eld instrument offersthe higher resolution that may be re-quired for the interference-free de-termination of sulfur or of the iso-tope ratios of some elements, e.g.,Cr, Fe, and V.19 An increase in res-olution inevitably leads, however, toa dramatic decrease in sensitivity. Itshould also be noted that the sensi-tivity of the latest generation quad-rupole instruments is only a factor of2–3 lower than that of high-resolu-tion ICP-MS operated in the low res-olution mode. A good tradeoff be-tween sensitivity, freedom from iso-baric interferences, and price is of-fered by ICP-MS instrumentsequipped with a collision cell thathave recently proliferated on themarket.20

Both quadrupole and sector-� eldmass spectrometers are scanning (se-quential) analyzers and multi-isotopeanalysis can be achieved at the ex-pense of the measurement sensitivityand precision. The sequential mea-surement of m /z values at differentpoints within a time-dependent con-centration pro� le of a transient sig-nal can result in peak distortions andquanti� cation errors, commonly re-ferred to as spectral skew.21 The al-ternative is TOF-MS, which featuresthe ability to produce a completeatomic mass spectrum in less than 50ms and thus allows the recording ofvery brief transient signals with high� delity.21 This is especially useful inthe on-line isotope ratio determina-tion, but a 10-fold loss in sensitivityof an ICP-TOF-MS instrument incomparison with the latest quadru-pole instruments may create an ob-stacle for the wider application ofICP-TOF-MS as a detector in thecapillary electrophoresis of metallo-biomolecules in biological systems.

The key to a succesful HPLC/CE–ICP-MS coupling is the interface.Figure 4 shows schematically themost frequently used combinations.In the simplest case, the exit of anHPLC column (i.d. 4.6–10 mm) isconnected to a conventional pneu-matic or cross� ow nebulizer. The useof capillary or megabore (0.32–1.0mm) HPLC systems, which are be-coming popular especially for re-versed-phase chromatography, re-quires the use of micronebulizers, ei-ther direct injection (DIN, DIHEN)or ones (e.g., Micromist) � tted witha small-volume nebulization cham-ber. The CE–ICP-MS coupling isless straightforward. The problemsdue to the laminar � ow generated bythe nebulizer suction, loss of sensi-tivity because of the electroosmotic� ow dilution by the makeup liquid,and peak broadening in the spraychamber have been resolved in thecommercially available inter facebased on a total-consumption self-aspirating micronebulizer � tted witha small-volume spray chamber.22,23

Figures 5–7 show three represen-tative examples of the use of HPLC/CE–ICP-MS in the speciation anal-ysis of metallobiomolecules. Size-exclusion chromatography coupledto ICP-MS allows the monitoring ofthe presence of stable metal com-plexes in liquid samples, e.g., tissuecytosols.24 The peak width is suf� -ciently large to allow the use of aquadrupole mass analyzer for the si-multaneous monitoring of up to 12isotopes. The correlation of the elu-tion volume with the molecularweight of the eluted molecule allowsthe determination of the molecularweight of the analyte. On this basis,a hypothesis regarding the identity ofthe eluted species can be put for-ward. Figure 5 presents a case inwhich the identity of a Pb–di-rham-nogalacturonane complex in wine iscon� rmed by the observed co-elu-tion of elements (Ba, Ce, Sr) with acharacteristic ionic radius � tting thecavity of the rhamnogalacturonanedimer.25

A � ner characterization of mix-tures of hydrophobic metal complex-es or of metal-containing protein


FIG. 4. Instrumental setups for HPLC and capillary electrophoresis with ICP-MS detection.

fractions isolated by size-exclusionchromatography can be achieved bycapillary/megabore reversed-phaseHPLC.26 Very sharp peaks can beobtained, as illustrated in Fig. 6,which shows the determination ofcyanocobalamin and its analoguesby HPLC–ICP-MS interfaced via adirect injection nebulizer. The thirdexample (Fig. 7) shows a mass � owCE–ICP-MS electropherogram of arabbit liver preparation of metallo-thionein.27 The individual isoformsare separated by capillary electro-phoresis, whereas S, Cd, and Zn arequanti� ed on-line by isotope-dilutionICP-MS. In this way, the stoichi-ometry of the metal–protein complexcan be determined.27,28

Electrospray-Ionization MassSpectrometry/Mass SpectrometryDetection in Chromatography andCapillary Electrophoresis. Theidenti� cation of unknown speciesdetected by ICP-MS can be achievedby running, in parallel, the same sep-aration using electrospray MS/MSdetection. In the standard instrumen-tal setup (Fig. 8), a low-� ow sepa-

ration technique, capillary HPLC orCE, is used. Another run is often car-ried out with a post-column acidi� -cation that allows the cleaving of themetal off the ligand and thus enablesthe direct determination of the mo-lecular weight of the latter.29 When acalibration standard of the analyte isavailable, a simple quadrupole massanalyzer is usually suf� cient for thecon� rmation of the analyte identity.A � ner characterization of unknownbioligands requires a tandem MS(e.g., triple quad or Q-TOF) or anMSn (e.g., ion-trap MS) instrument.

Figure 9 shows an example of theidenti� cation of a metal-b indingpolypeptide and its complexes withCd 21 by reversed-phase HPLC–ESI-MS. In ESI-MS, proteins (and pro-tein–metal complexes) show a char-acteristic envelope of peaks due tomultiple ionization that allows theaccurate (60.5 Da) determination ofthe molecular weight (Fig. 9a). Themass spectrum taken at the apex ofa peak during chromatography ofrabbit liver metallothionein prepara-tion (Fig. 9b) allows the determina-

tion of the molecular weight of themetal–metallothionein complex.30 An-other mass spectrum (Fig. 9c), ob-tained after on-line acidi� cation ofthe chromatographic ef� uent, allowsthe determination of the molecularmass of the apo- (metal-free) metal-lothionein and thus allows a searchfor its identity in a database. 29 Thedifference between the M r of thecomplex and M r of the ligand is ameasure of the complex stoichiom-etry and can be used for the valida-tion of data obtained in the experi-ment outlined in Fig. 7.

Another example (Fig. 10) showsa � ne characterization by CE–ESI-MS/MS of a Cd containing fraction(isolated by size-exclusion chroma-tography (SEC)) of the cytosol ofsoybean cells exposed to a stress ofCd 21.31 The total ion-current electro-pherogram shows a number of peaksto which a molecular mass value canbe attributed. The identi� cation ofthe compounds, in this case a seriesof polypeptides referred to as phy-tochelatins, is possible by acquiringon-line MS/MS data. The interpre-


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FIG. 5. Speciation analysis of trace elements in wine by size-exclusion chromatogra-phy with multielement detection by ICP-MS.25 The arrows indicate elution of the molec-ular weight markers (pollulans) used to calibrate the column. The hydrodynamic vol-ume and the coelution of Ba, Ce, Pb, and Sr suggest the elution of a metal complexwith the dimer of rhamnogalacturonan (partly shown in the inset).

FIG. 6. Reversed-phase HPLC–ICP-MS chromatogram of cyanocobalamin and its ana-logues obtained using a microbore C8 column coupled to ICP-MS via a direct injectionnebulizer.32

tation of the CID mass spectrum ofthe protonated molecular ion respon-sible for a peak in the CE–ESI elec-tropherogram allows the determina-tion of the amino acid sequence ofeach of the separated polypeptides.

Multidimensional HyphenatedTechniques in Speciation Analysis.The complexity of the biological ma-trix may require the combination oftwo or more separation mechanismsin series to assure that a unique met-al species arrives at the detector at agiven time. This approach is illus-trated in Fig. 11, which shows an ex-ample of the identi� cation of an ar-senic species in a marine biota. Theindividual arsenic compounds areisolated by tri-dimensional LC in-cluding size-exclusion, an ion-ex-change, and cation-exchange mech-anisms. The isolated species are an-alyzed by electrospray TOF-MS; theaccuracy of the molecular mass mea-surement (especially important in thecase of compounds of As, which isa monoisotopic element) and thematching of the isotopic pattern ofthe molecular ion allow the identi� -cation of the compound. The identitycon� rmation or identi� cation of anunknown compound can be achievedby CID-MS.

MOLECULAR DESCRIPTIONOF MECHANISMS OF TRACEELEMENT INVOLVEMENT INBIOLOGICAL SYSTEMS

Hyphenated techniques allow theidenti� cation in a biological sampleof a metal-complexing metabolitecoded directly by a gene (e.g., me-tallothioneins), or biosynthesized bya metal-activated enzyme coded by agene (e.g., phytochelatins, citricacid, nicotianamine) (see, for in-stance, Fig. 1). In this context, therole of molecular biology techniquesfor the isolation and cloning of thecorresponding gene is rapidly grow-ing in importance. The identi� cationof the metabolite (metal-binding li-gand) as functionally important, e.g.,in a hyperaccumulating plant, shouldbe accompanied by the identi� cationof the relevant gene, its cloning, andits 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 metal-lothionein preparation obtained using the CETAC CEI-100 interface and quanti�cationby isotope dilution.27 The quantities given refer to the amount of protein producing thecorresponding peak. The spatial structure of the molecule, eluting as the peak high-lighted in yellow, is shown in the inset of the �gure.

FIG. 8. Instrumental setups for HPLC and CE with electrospray MS and MS/MS detection.

of an identi� cation achieved by a hy-phenated technique may thereforeonly be considered validated if thegenetically modi� ed bacteria oryeast exposed to metal stress willbiosynthesize the same metallocom-pound as the original plant or ani-mal.

Note also that the isolation, clon-ing, and expression of functionalgenes may be considered as an ad-vanced sample preparation methodfor speciation analysis. It allows theexchange of the complex matrix of aplant or animal cytosol into a simplermatrix of bacteria or yeast extractwith the simultaneous enrichment ofthe metallospecies of interest. In thisway, a hyphenated technique will beused to con� rm the presence of anexpected species rather than to iden-tify it among thousands of other bio-ligands.

CONCLUSION

The decreasing detection limits ofICP-MS, the availability of ef� cientinterfaces to HPLC and capillary


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FIG. 9. Determination of the molecular mass and the degree of metallation of metallo-thioneins by reversed-phase HPLC with electrospray MS detection.29 The inset (a) givesthe principle of the molecular mass determination of a protein and of its complex witha metal. Insets (b) and (c) give mass spectra obtained at the apex of peak 4 of thechromatogram in a setup without and with post-column acidi�cation, respectively.

FIG. 10. CE–ESI-MS/MS characterization of polypeptides in a Cd containing fractionisolated from the cytosol of soybean cells by size-exclusion chromatography.31 A totalion current CE–ESI-MS electropherogram is shown. The mass spectrum at the apex ofa peak allows the determination of the molecular weight of the peptide present. Theelectropherogram acquired using CID of the protonated ions of the molecules found byCE–ESI allows the determination of the amino acid sequence of the peptides detected.An example structure of the major compound found (/b-alanine-PC3) is shown.

electrophoresis, and the increasingsensitivity of electrospray MS formolecule-speci� c detection at tracelevels open the way to the character-ization of endogenous trace elementspecies in biological systems and of-fer the key to the understanding ofmany chemical processes essentialfor life. The development of analyt-ical methods for biochemical speci-ation analysis is being carried out atthe crossroads of interest of manydisciplines and can pro� t from theinterdisciplinarity of approach to thesame degree that it can suffer fromthe lack of it. In order to cope withthe complexity of biological matri-ces, multidimensional separation anddetection approaches are required.The elucidation of the role of a metalspecies identi� ed by multidimen-sional analytical techniques in a bi-ological system requires the identi-� cation of the gene coding the cor-responding bioligand directly, orcoding an enzyme responsible for itssynthesis, the isolation of the gene,and its expression in a bacteria oryeast. A positive identi� cation of themetabolite, previously identi� ed inthe original plant or animal tissue, inthe mutant bacteria or the yeast, willdeliver the ultimate proof of its in-volvement in the metal metabolism.The more powerful analytical tech-niques make the question about thepreservation of the original formmore pertinent than ever; direct spe-ciation analysis of liquid sampledfrom an individual cell appears as along-term goal.

ABBREVIATIONS

AES atomic emission spec-trometry

TOF time-of-� ightCE capillary electrophoresisCEC capillary electrochroma-

tographyCID collision-induced disso-

ciationDIN direct-injection nebuliz-

erDIHEN direct-injection high-ef-

� ciency nebulizerESI electrospray ionizationFT-ICR Fourier transform ion

cyclotron resonance


FIG. 11. Identi�cation of an arsenosugar by multidimensional hyphenated techniques. (a) Puri�cation of the arsenic compound: theAs-containing fraction from SEC was puri�ed by anion-exchange chromatography. Each of the anion-exchange chromatographicfractions was further puri�ed by cation-exchange HPLC. Arsenospecies eluted from the cation-exchange column were analyzed byESI-TOF-MS; (b) electrospray TOF mass spectra: the vicinity of a peak corresponding to an arsenic-containing compound waszoomed to reveal the isotopic pattern. The error of the Mr determination represents the difference between the calculated and mea-sured molecular mass values; (c) CID-TOF mass spectrum of the protonated ion of the organoarsenic compound isolated by a quad-rupole �lter. The structure correponds to an arsenosugar: 3-[59-deoxy-59-(dimethylarsinoyl-b-ribofuranosyloxy]-2-hydroxypropyleneglycol.


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HPLC high-performance liquidchromatography

ID isotope dilutionICP inductively coupled

plasmaMC multicollectorMEKC micellar electrokinetic

chromatographyMT metallothioneinPAGE polyacrylamide gel elec-

trophoresisQ quadrupole used as a

mass analyzerq quadrupole used as a

collision cellSDS sodium dodecyl sulfo-

nateSF sector � eld

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Hyphenated Techniques for Elemental Speciation in Biological Systems