biochemical speciation analysis by hyphenated techniques

Analytica Chimica Acta 400 (1999) 321–332

Biochemical speciation analysis by hyphenated techniques

Ryszard Łobinskia,b,∗, Joanna Szpunara

a CNRS EP 132, Hélioparc-Pyrénées, 2, av. Pr. Angot, 64053 Pau Cédex 9, Franceb Department of Chemistry, Warsaw University of Technology, ul. Noakowskiego 3, 00-664 Warszawa, Poland

Received 15 April 1999; accepted 13 July 1999

Abstract

The elucidation of mechanisms that govern the essentiality and toxicity of trace elements in living organisms is criticallydependent upon the possibility of the identification, characterization and determination of chemical forms of these elementsinvolved in life processes. The recent progress and the state-of-the-art of biochemical species-selective trace element analysisare critically evaluated with particular emphasis on the use of techniques combining the high selectivity of high performanceliquid chromatography (HPLC) with the elemental or molecular specificity of mass spectrometry [using inductively coupledplasma (ICP) or electrospray ionization (ESI)]. The potential and limitations of hyphenated techniques as a tool for speciationof metals and metalloids in biological materials is discussed using a number of examples drawn from the latest research inthe authors’ laboratory. ©1999 Elsevier Science B.V. All rights reserved.

Keywords:Speciation; Bio-inorganic chemistry; Chromatography; ICP MS; Electrospray MS/MS; Selenium; Phytochelatins; Metallothioneins

1. Introduction

The proper functioning of life is critically depen-dent on trace elements in a number of different ways.Some trace elements are highly toxic whereas others,considered essential, are needed for the accomplish-ment of life processes. A surge of evidence during thepast 20 years has been leading to the conclusion that itis not the total element content but that of a particularspecies that should be determined if valid informationon the essentiality or toxicity of a given element is tobe obtained [1–4].

IUPAC defines chemical species as a specific andunique molecular, electronic, or nuclear structure ofan element. Speciation of an individual element refers

∗ Corresponding author. Fax: +33-5-59-80-68-85E-mail address:[email protected] (R. Łobinski)

to its occurrence in or distribution among differentspecies. Speciation analysis is the analytical activityof identifying and quantifying one or more chemicalspecies of an element present in a sample [5]. Theunderstanding of molecular mechanisms of metals es-sentiality and toxicity in biochemistry is critically de-pendent on the analytical techniques able to provideinformation on the chemical forms in which elementsare present in living organisms. Speciation analysishas been one of the fastest developing areas of analyt-ical chemistry towards the close of the 20th century[6–8]. The areas targeted and the species of concernare summarized in Fig. 1.

Speciation-related activities continually reported inthe literature can be considered from the scientificpoint of view at three different levels: (i) demonstra-tion of analytical craft and skills of an analyst to de-termine a particular elemental species in a sample;

0003-2670/99/$ – see front matter ©1999 Elsevier Science B.V. All rights reserved.PII: S0003-2670(99)00628-5

322 R. Łobinski, J. Szpunar / Analytica Chimica Acta 400 (1999) 321–332

Fig. 1. Target areas and species of concern in speciation-related research.

(ii) exploratory investigations aimed at the detectionof unknown elemental species in a tissue of a livingorganism, their identification and/or structural char-acterization, and (iii) use of the acquired informationto understand the mechanisms governing transforma-tions of trace elements in biological systems.

The analytical craft and skills have usually beendemonstrated for the determination of anthropogenicorganometallic compounds and products of their en-vironmental degradation: methylmercury, alkylleadcompounds, butyl- and phenyltin compounds, andsimple organoarsenic and selenium species [7,8].Since the compounds of concern are mostly anthro-pogenic, standards are available or can be fairlyreadily synthesized. Why, this is the availability ofthe standards that is often the motive force for therealization of a work. This area, since the pioneeringwork of Van Loon [9], has been dominated by atomicspectroscopists for whom chromatography has be-come an attractive sample introduction technique thatopened the way to sensitive species-selective anal-

ysis. The works have focussed on developments ofinstrumentation and sample preparation procedureswhile the major challenges included recoveries, pre-cision, accuracy, harmonization of measurements andnormalization of methodology with the culminationby production of certified reference materials [8].

The aim of exploratory investigations of elemen-tal species in biological samples has been the questfor knowledge on the speciation of a given elementin a living organism. The validity of the natural prod-ucts chemist’s approach including isolation of a suf-ficient quantity of purified species for NMR studiesdemonstrated for organoarsenic compounds [10]. Thenon-availability of standards and the need for prelim-inary knowledge on compounds to be looked for havebeen keeping an analytical chemist away. The poorstability of many species of potential interest, insuf-ficient detection figures of merits of analytical tech-niques for the molecular characterization ESI MS andNMR, and the need for complex multistep separationtechniques for purification of tiny amounts of species

R. Łobinski, J. Szpunar / Analytica Chimica Acta 400 (1999) 321–332 323

prevented this area from a rapid progress despite theneed for information from biochemists and bioinor-ganic chemists.

Information obtained by the analyst should respondto the following questions:1. Role of essential and beneficial trace elements in

life processes, such as metal ion transport or en-zyme activation

2. Metabolism of trace elements: formation of com-pounds with the true carbon metalloid bond suchas organoselenium and organoarsenic compounds,biosynthesis of ligands in response to the metalstress (phytochelatins, metallothioneins) and com-plexation of toxic ions.

3. Action of mutagenic and carcinogenic elementsin living organisms: molecular targets and mech-anisms of action.

The revival of interest of trace element analyticalchemists in biochemistry has been observed sincethe advent of ICP MS and the relatively straightfor-ward on-line coupling of these technique with highresolution liquid chromatography and capillary zoneelectrophoresis [6,11,12]. Indeed, ICP MS and highresolution ICP MS have been offering previouslyunheard-of sensitivities to monitor selectively in realtime a given element as part of a compound exiting achromatographic column or an electrophoretic capil-lary. A 30 min chromatographic run usually allows theidentification of the presence of a number of thermo-dynamically stable and kinetically inert species of anelement in the sample. An urgent issue is becomingthe identification of signals in the increasing numberof acquired chromatograms for different biologicalsamples. Recent successful applications of electro-spray mass spectrometry (ESI MS) for this purposeconfirm the exciting potential opportunities offeredby this technique [13,14].

The objective of this paper is to present thestate-of-the-art of a fairly young discipline: bio-inorganic analytical chemistry which is likely todominate trace element analysis in the next century.Hyphenated techniques seem to be the key to theworld of information on the identity and concentra-tions of trace elements species in plants, animal andman. The exploration of this world is the prerequi-site to understand the role of trace elements in lifechemistry.

2. Elemental species in living organisms

The millions of year of evolution of biological sys-tems resulted in the great variety of trace elementspecies present in biological systems. From the chem-ical point of view they can be divided in several cate-gories [1,15,16]:(a) biosynthesized molecules with the ‘true’ metal(metalloid)–carbon bond. This category includesselenoaminoacids and their higher analogues: se-lenoglutathione and selenoproteins. They can co-ordinate metals, especially Hg, using the Se atomas the coordination center. Another important classincludes organoarsenic compounds, e.g. arsenobe-taine and arsenosugars.

(b) complexes with amino acids, oligopeptides andpolypeptides (proteins). Metal complexes with pro-teins, including enzymes, are carriers of biochem-ical function. Whereas the carboxamide functionitself of peptide bonds –C(=O)–N(–H)– is only apoor metal coordination site, peptides contain sev-eral functional groups in the side chains that are par-ticularly well suited for metal coordination. Theyinclude especially cystein (–CH2SH) and methio-nine –CH2CH2SCH3 which bind metals with sul-fur affinity (Cd, Cu, Zn) in compounds such asglutathione, phytochelatines, and metallothioneins;and histidine of which both nitrogen atoms becomeavailable for coordination after metal-induced de-protonation (e.g. Cu, Zn in superoxide dismutase).

(c) complexes with nucleobases, oligo- and polynu-cleotides, and -nucleosides. Heterocyclic nucle-obases, alone or as constituents of nucleosidesor nucleotides offer several different coordinationsites for metal ions. Of particular interest is the co-ordination of metal ions, e.g. CrO4− or inert metalcomplexes to DNA because of the specificity withregard to certain base-pair sequences in the doublehelix.

(d) complexes with biosynthesized macrocyclicchelating agents. The most important group is theanalogues of tetrapyrrole which in their deproto-nated form can tightly bind even relatively labiledivalent metal cations. The best known compoundsof this group include chlorophyll and productsof its degradation, cobalamins (the coenzymati-cally active forms of Vitamin B12), and porphyrins


including the heme group found in haemoglobin,myoglobin, cytochromes and peroxidases.

(e) complexes with other biomacromolecules(polysaccharides, glycoproteins). Relatively littleis known about the relevance of metal coordinationto lipids and carbohydrates, although the poten-tially negatively charged oxygen functions can bindcations electrostatically and even undergo chelatecoordination via polyhydroxy groups.

3. Hyphenated techniques – state of the art

The recent progress in microscale isolation and pu-rification techniques, such as, especially HPLC, CZEand capillary electrochromatography, on one hand, andthe increasing sensitivity of trace analysis by ICP MSon the other hand, were at the origin of a new genera-tion of analytical methodology based on the couplingof a high performance separation technique with an ul-trasensitive atomic spectrometric detector [4,6,11,12].This analytical approach is becoming a fundamentaltool for the functional characterization of trace el-ements or otherwise unaccounted for metal ions inbiological systems. As calibration standards are un-available for most of trace element species in liv-ing organisms, the use of molecule specific (and notelement-selective) detection technique, such as ESIMS or NMR is necessary to complete the character-ization of the species detected by HPLC–ICP MS.The most frequently used hyphenated techniques areschematically presented in Fig. 2.

3.1. Amenability of a hyphenated technique tobiochemical speciation analysis

The prerequisite for a hyphenated technique to beapplied to speciation analysis is that the elementalspecies injected on a chromatographic column leavesthe column unchanged, and that no artefact species(absent in the original sample) are generated on thecolumn. This implies a fairly high thermodynamic sta-bility and kinetic inertness of the species of interest.The metal–ligand bond should be much stronger thatthe interaction either of the metal or of the ligand withthe column stationary phase. Otherwise a risk occursthat the metal complex will be destroyed by sorption(ion-exchange) of the metal (or of the ligand) on the

Fig. 2. Schematic representation of frequently used hyphenatedtechniques for speciation analysis.

column material or that metal ions sorbed on the col-umn will be complexed by a free ligand in the injectedsolution generating a species that had not existed inthe sample.

The mobile phase used should imitate in terms ofpH and composition the sample matrix which may notallow the separation of the species of interest. Columnstationary or mobile phases competing with or displac-ing ligands from the analyte metal complex should beavoided. The minute amounts of the sought-for speciesare present in complex matrices. The simplification ofthe matrix often changes the identity of the species.

The success of an analytical speciation approachcritically depends on three other factors(i) Sensitivity of the chromatographic detector. Thetrace element present in a sample is fractionated intodifferent species so at a given time only a small frac-tion of its total concentration arrives at the detector.Consequently, sub-picogram detection limits are re-quired to assure a successful speciation analysis oftrace elements in body fluids or tissue homogenates.This requirement favours the currently most sen-sitive spectrometric techniques for elemental anal-ysis: ICP MS and high resolution ICP MS. Forsome elements exogenous contamination is likelyand blank problems are omnipresent.

(ii) Separation between different species of the ele-ment. The chromatographic technique used shouldguarantee that the signal corresponds to one par-ticular species. Since biological tissues are verycomplex mixtures containing thousands of differ-


ent compounds, the use of several successive sepa-ration techniques with different separation mecha-nisms may be required.

(iii) Isolation of a sufficient quantity of elementalspecies so that its identity could be confirmed bya technique offering the molecular selectivity, e.g.ESI MS/MS or NMR.

3.2. Detection of trace element species in biologicalsystems by size exlusion chromatography–ICP MS

The detection of a metal compound in a sampleis the prerequisite of any further action concerningits identification, characterization and the role in bio-chemistry. The actual problem can be reduced to prov-ing that the analytical signal detected by the spec-trometer is due to the presence of a metal compoundand not to a simple metal ion. This can be done bypreceding the detection by a separation technique thatwould differentiate between the free metal (or metal-loid) ion and the same element bound with a larger,usually macromolecular structure.

The detection of the presence of metal complexeswith biological macromolecules has commonly beenrealised by ultrafiltration using a filter with a smallcutoff molecular mass, usually 500–5000 Da. The con-centration of the element of interest was determinedin the initial sample and in the filtrate, usually bygraphite furnace AAS; the metal retained was con-sidered to be bound to a macromolecular species. Arefinement of this technique by the successive ultra-filtration through membranes with molecular weightcut-offs of 500, 5000 and 30 000 Da has been widelyused to study the distribution of metal-species as afunction of the molecular weight. The method allowsonly a rough speciation and is time consuming.

An improvement in terms of resolution can be ob-tained by the use of size-exclusion chromatography(SEC). This technique is less cumbersome than ultra-filtration and is also faster since it allows the on-linedetection. The coupling SEC–ICP MS is the most pop-ular technique for the first screening of an unknownsample in view of the presence of macromolecularspecies of elements [17]. It should be noted that theseparation mechanism is never based uniquely on themolecule size and a reasonable degree of separationcan be obtained by SEC for small organometalloid

molecules, e.g. organoselenium compounds, which donot differ significantly in terms of molecular weight[18].

3.3. Verification of the chromatographic purity –orthogonal separation mechanisms: ion-exchange,reversed phase HPLC and CZE

Biological fluids and tissue supernatants are verycomplex mixtures containing thousands of differentcompounds. Each of the fractions eluting from a SECcolumn is still a very complex mixture. To associateunequivocally a particular trace element with a bio-compound in a given fraction is not so evident. A fur-ther purification of the metallocompound, by a separa-tion technique using a different separation mechanism,e.g. by hydrophobicity or electrical charge is neces-sary. A difficulty is that the element at trace and ultra-trace levels can be detected easily by ICP MS but thisis not the case of the ligand. On-line UV–VIS spec-trometry for species with chromophoric groups (e.g.proteins), or refractometry for polysaccharides, needto used despite their limited sensitivity and selectivity.The chromatograms are complex and the associationbetween the bioligand and the trace element made isoften speculative to a large degree.

When the species of interest is sufficiently stable,it is recommended to heartcut the metal-containingfraction and to verify its purity in terms of the num-ber of metal-containing species present. This can bedone for example by ion-exchange or reversed-phasechromatography while monitoring the elution of met-allocompounds with ICP MS. In the case of complexsystems, CZE owing to its high resolution on the peakheartcut by chromatography seems to be the final toolfor verification of the complexity of trace element spe-ciation.

Affinity chromatography is recommended for thepurification of metal complexes with protein. If thetrace element still sticks to that protein bound to theantigen on the column, the linkage between the twobecome convincing [2].

3.4. Identification of the species of interest

The lack of convenient methods of structure deter-mination and confirmation remains a major barrier to


the mechanistic understanding of functions, toxicityand bioavailability of elemental species in the chem-istry of life. Access to structural information for theidentification of known, unknown or unexpected com-pounds is a great challenge to biochemical speciationanalysis, especially that the improving sensitivity ofICP MS instruments will inevitably increase the num-ber of species detected.

The simplest method is comparing the retentiontime of the species detected with that of a standard.This approach, which is fairly successful for anthro-pogenic organometallic species, usually fails in thebiochemical speciation analysis. Standards are usu-ally not available since the majority of species presenthave not been discovered or identified yet. Also,since, as discussed about, the samples are extremelycomplex in terms of the number of ligands present,there is a probability that several species can havethe same retention time on some chromatographiccolumns. Therefore, analytical techniques that offerthe possibility of the identification and characteriza-tion of the metal complex or at least of the ligand,such as molecular mass spectrometry, now usually us-ing the electrospray ionization (ESI MS), or NMR arenecessary.

The application of ESI MS to the identification ofmetallocompounds detected in body fluids and tissueextracts by ICP MS suffers from two major shortcom-ings. The first is the discrepancy between the detec-tion limits of ESI MS (10–100 ng ml−1) and those ofICP MS (0.1 ng ml−1). The second is the vulnerabilityof ESI MS (even in the pneumatically assisted mode)to the presence of salts that suppress the ionization ofthe analyte compounds and reduce the signal inten-sity. Due to of these two reasons no success could beachieved with the identification of metallocompoundsby running HPLC–ESI MS or CZE–ESI MS in parallelin the same conditions as HPLC–ICP MS or CZE–ICPMS. The problem of insufficient quantity of an ele-mental species to be identified available is even muchmore pertinent in NMR.

A way to overcome these shortcomings iselimination of salts by running chromatography(size-exclusion or reversed-phase) with salt-freebuffers, heart-cutting of the fraction containing theto-be-identified species, and evaporative preconcen-tration (freeze-drying) followed by ESI MS or ESIMS/MS.

4. Applications of hyphenated techniques tobiochemical speciation analysis

4.1. Speciation of biosynthesized organometalloidcompounds: selenium in yeast

4.1.1. RationaleSelenium is an essential micronutrient for living

organisms but may be toxic in specific forms [e.g.Se(VI)] and at higher concentrations [19]. It is a com-ponent of the human enzyme glutathione peroxidaseand has been reported to show a protective effectagainst cancer [20]. Se-enriched yeast is a popularfood supplement. Since the bioavailability and the tox-icity of Se are closely correlated with its chemicalform, there has been increased interest recently in se-lenium speciation in yeast [13,18,21–23].

4.1.2. Detection of organoselenium compounds inyeast extracts

Fig. 3a shows a chromatogram obtained bySEC–ICP MS of an aqueous extract of a selenizedyeast sample obtained as described elsewhere [13,18].Despite the fact that, in theory, the separation shouldbe based on the analyte molecular weight, secondaryadsorption and ion-exchange effects play apparentlyan important role in SEC. At the concentration levelsinvolved, a. 0.1mg ml−1, no identification of the shad-owed peak is possible by any on-line technique. Sincethe elution volume of the peak does not correspond tothat of any of the standards available: Se(IV), Se(VI),selenomethionine, selenocysteine, selenoethionine theonly possibility is the heart-cutting of the peak, evap-orative preconcentration and examination of the solu-tion obtained by ESI MS. Note that the concentrationlevel is too low for NMR to be successful.

4.1.3. Identification of the compound detected byESI MS

Fig. 3b shows an ESI mass spectrum of a solutionobtained by heartcutting of the shadowed peak in Fig.3a. An ion-cluster centred atm/z433 with a character-istic natural abundance Se isotope pattern can clearlybe seen. It corresponds to the molecular ion cluster ofa protonated80Se-compound with a molecular mass432.0. No other cluster showing a similar patterncan be seen which indicates that the isolated fraction


Fig. 3. Identification of a selenocompound in selenized yeast [13]. (a) size-exclusion HPLC chromatogram with ICP MS detection ofan aqueous extract of selenized-yeast prepared as described elsewhere [18]. The heart-cut fraction was shadowed; (b) electrospray MSspectrum of the shadowed fraction after evaporative preconcentration. The part of the spectrum containing the Se-characteristic isotopepattern is zoomed in the inset; (c) collision induced dissociation mass spectrum (product ion scan) of the80Se-containing molecular ion(433.0 u). The fragmentation pattern of the identified compound: Se-adenosyl-homocysteine is showed in the inset.


contains probably only one selenium species. Themolecular mass may be an indication of the speciesidentity but a lot of preliminary information on thesystem investigated is necessary to put forward a hy-pothesis on the identity of the compound. A deeperinsight into this identity can be obtained by frag-menting the molecular peak by a collision induceddissociation (CID) process.

4.1.4. Characterization of the selenocompoundsdetected by ESI tandem MS

The CID mass spectra (Fig. 3c) of ions withm/z433 and 431 u corresponding to the two most abun-dant selenium isotopes:80Se and78Se allow the dif-ferentiation between fragments that contain selenium(the m/z signals in both spectra appear at a differ-ence of 2 u) and fragments that do not contain sele-nium (them/z signals in both spectra have the samevalue). In this way two80Se-containing fragments withmasses of 182.0 and 298.0 u can be identified in ad-dition to the molecular ion (433.2 u) that obviouslycontains selenium. The fragments that do not containselenium can be seen as peaks at 136.0 u and a peakat 250.0 u. The fragmentation pattern shown in theinset of Fig. 4 corresponds with a great probabilityto Se-adenosyl-homoselenocysteine. This compoundwas reported in the literature in the context of the trans-methylation and polyamine synthesis pathway in themetabolism of selenomethionine in mammals [24].

4.2. Characterization of metallothionein–cadmiumcomplexes in animal tissues

4.2.1. RationaleSequestration of metal ions (e.g. Cd2+) in stable,

intracellular macromolecular complexes is a majormechanism by which cells resist the cytotoxic ef-fects of the metal [25–27]. Metallothioneins (MTs) [agroup of non-enzymatic low molecular mass proteins(6–7 kDa)] represent a class of the most commonligands [25–27]. Classic techniques for the deter-mination of MTs such as metal-saturation assays,polarography via sulfhydryl groups and immunoas-says fail to provide information on the original metalcomposition and do not allow one to identify and toquantify the individual MT isoforms. The complexityof the polymorphism of mammalian metallothioneins

and the variety of potentially bound metals (Cd, Zn,Cu, Hg) requires an approach coupling a high resolu-tion separation technique able to distinguish betweenthe proteins with a single amino acid heterogeneity,and an element-selective detection technique able todetermine the metals bound [28].

4.2.2. Detection of stable Cd complexes in tissuecytosols

Fig. 4a shows a SEC–ICP MS chromatogram ob-tained for a preparation of rabbit liver metallothionein.It shows two peaks: one (shadowed) at an appar-ent molecular weight of 12 kDa corresponds to theMT-bound cadmium. The other: close to the total vol-ume of the column corresponds to Cd2+ complexedwith b-mercaptomethanol added to the sample. Cd2+is not eluted from the column as a cation because ofthe retention on the column stationary phase.

4.2.3. Determination of the chromatographic purityof the SEC–ICP MS signals

The shadowed fraction heart-cut, lyophilized anddissolved in a minimum quantity of water turns outto contain at least two species that can be separatedon an anion-exchange column (Fig. 4b). The peakscorrespond to two MT-isoforms denoted as MT-1 andMT-2 according to their order of elution [29]. A differ-ent separation mechanism, e.g. reversed-phase chro-matography (Fig. 4c) applied to the heart-cut MT-2fraction shows that this peaks has attained a certaindegree of purity. There is still, however, no guaran-tee, that the signal corresponds to a pure compound,neither any clue regarding the identity of these com-pounds (tentatively termed as sub-isoforms) [30]. Notethat the stability of Cd–MT complexes allows the sepa-ration of intact complexes with different mobile phasecomposition in the pH range 6–8.6 using different sta-tionary phases.

4.2.4. Signal identification by electrospray massspectrometry

The key to verify the purity of the eluted fractionand to determine the identity of the compound elutedis the application of a detector specific of the moleculeand not of the metal. Despite some success reportedrecently with controlled fragmentation in low-powerICPs [31], electrospray MS remains the best suited


Fig. 4. Identification of metallothionein isoforms in an animal tissue. (a) size-exclusion chromatogram with ICP MS detection; (b)anion-exchange chromatogram with ICP MS detection; (c) microbore reversed-phase chromatogram with ICP MS detection via a directinjection nebulizer; (d) microbore reversed-phase chromatogram with ESI MS detection (total ion current); (e) mass spectrum taken at theapex of the major chromatographic peak in Fig. 4d (pH 6.0); (f) mass spectrum taken at the apex of the same peak after post-columnacidification (pH 2.0).

technique for the on-line determination of molecularmass of the MT-complexes eluting from a chromato-graphic column [32,33] or from an electrophoreticcapillary [34]. Fig. 4d shows a reversed-phase chro-

matogram with electrospray MS detection (totalion count) corresponding to the chromatogram withcadmium-selective spectrum by ICP MS shown inFig. 4c. The mass spectrum taken at the apex of


the peak (snapshot) shows that despite the Gaussianshape this compound contains a number of speciesthat can be differentiated by the molecular mass.

A deeper insight into the identity of the eluted com-plex can be gained by on-line dissociation of the com-plex (e.g. by acidification) and identification of the lig-and. Fig. 4e shows that the mass spectrum of the lig-and is much simpler than that of the complex and con-tains one major compound having a molecular weightof 6125.5± 0.5 u that can be identified as the MT-2asub-isoform. The complexity of the mass spectrum inFig. 4d results from the fact that it contains the differ-ently metallated (mixed metal complexes) of the sameligand (MT-2a). The difference between the molecu-lar mass of the complex and that of the ligand (bothdetermined by ESI MS) allows the calculation of thecomposition of the eluted species.

4.3. Characterization of phytochelatin–cadmiumcomplexes

4.3.1. RationalePhytochelatins (PCs) are a class of peptides com-

posed only of three amino acids: cysteine (C), glu-tamic acid (E) and glycine (G) and in which glutamicacid is linked to cysteine through ag-peptide linkage.Their general formula is (EC)nG wheren is comprisedbetween 2 and 11 [35,36]. They are synthesized fromglutathione (GSH) during a reaction catalysed by anenzyme called PC-synthase in the presence of someheavy metals which procures plant a certain degreeof tolerance to the heavy metal stress by complexedthrough the cysteine sulfur atom [35,36]. The PCs gen-eral structure is conservative in a wide variety of plantsbut some modifications may occur on the C terminalamino acid. (EC)nA has been detected in vegetables.(EC)nS, (EC)nE and (EC)n have also been underscoredrespectively in rice, in maize and in yeast. These mod-ified PCs are named iso-PCs.

4.3.2. Screening for the presence of phytochelatins bysize-exclusion chromatography with ICP MS detection

Similarly as in the above discussed case of metal-lothioneins, the first step is the detection of the pres-ence of stable metal-species in an aqueous extract ofa plant material to be investigated. This can be read-ily carried out by the coupling of size-exclusion chro-

matography and ICP MS [37]. Fig. 5a shows a typicalchromatogram for a plant that had been exposed to theCd stress. The sample chromatographed is an aqueousextract of such a sample. The chromatogram shows anumber of Cd-species present without giving any in-dication in terms of their identity. The mass balanceof Cd indicates that more than 50% of Cd present inthe extract leaves the column in the form of Cd com-plexes.

4.3.3. Identification of Cd-complexes by reversedphase HPLC with ESI MS detection

Contrary to the above discussed case of metal-lothioneins no success could be obtained with thechromatography of the extract using a different sep-aration mechanism. Reversed-phase chromatographywith a neutral mobile phase necessary to preservethe integrity of the complex (data not shown) leadsto the presence of a single intense signal of Cd inthe void of the column. The identification of theeluted compounds can be approached, however, byreversed-phase chromatography in acid media [38].Since the complex is likely to be dissociated in acidconditions, the detection technique should target theligand. ESI MS is a convenient detector for this pur-pose as demonstrated in Fig. 5b. The total ion currentchromatogram shows a number of species present inthe analysed solution. The molecular mass of an elut-ing compound can be determined by acquiring a massspectrum at the apex of the chromatographic peak.Using this method the most intense signal was foundto correspond to a compound with the molecularweight equal to that of PC3 (Fig. 5c).

4.3.4. Sequencing of phytochelatins by ESI MS/MSAn unambiguous confirmation of the identity of the

eluted species can be obtained by fragmentation of themolecular ion of the species to be identified. Peptidesare known to fragment primarily at the amine bondsto produce a ladder of sequence ions [39]. The chargecan be retained on the amino terminus (Type b ion)or on the carboxy terminus (Type y ion). Thus a com-plete series made of ions from both types allows theamino acids sequence determination by subtraction ofthe masses of adjacent sequence ions. This character-istic fragmentation pattern allows in protoneted prac-tice sequencing of peptide ligands (up to ca. 2000 Da)


Fig. 5. Identification of phytochelatins in water extracts of plants exposed to the cadmium stress. (a) size-exclusion HPLC chromatogramwith ICP MS selective detection of Cd (pH 8.5); (b) reversed-phase chromatogram (acidic conditions) with ESI MS detection (total ioncurrent) of the phytochelatin fraction in a plant extract preconcentrated by lyophilization; (c) mass spectrum taken at the apex of the peakshadowed in Fig. 5b; (d) collision induced dissociation mass spectrum of the most intense (molecular) peak in Fig. 5c.

bonding metals by using collision induced dissociationof the peptide protonated molecule ion. Fig. 5d showsa mass spectrum (product ion scan) of the molecularion of the investigated species that confirms the aminoacid sequence of PC3.

5. Conclusions

Bioinorganic analytical chemistry is a rapidly de-veloping field of research at the interface of trace ele-ment analysis and analytical biochemistry which tar-


gets the detection, identification and characterizationof complexes of metals (metalloids) with moleculesof natural origin (biomolecules) by coupled (hyphen-ated) techniques. Hyphenated techniques based on thecoupling of a highly selective high performance liq-uid chromatography (HPLC) or capillary zone elec-trophoresis (CZE) with the elemental or molecularspecific mass spectrometry [using inductively coupledplasma (ICP) or electrospray ionization (ESI)] are be-coming a fundamental tool for the functional charac-terization of trace elements or otherwise unaccountedfor metal ions in biological systems. In addition tofurther improvements in the sensitivity and toleranceto matrix of ESI MS, a major challenge remains thedevelopment of custom-designed sample preparationprocedures allowing biochemical speciation in solidmaterials.

References

[1] R. Lobinski (Ed.), Metals and Biomolecules, AnalusisMagazine, 26 (1998) M19.

[2] R. Cornelis, J. De Kimpe, X. Zhang, Spectrochim. Acta 53(1998) 187.

[3] A. Sanz Medel, Spectrochim. Acta 53 (1998) 197.[4] J. Szpunar, R. Lobinski, Pure Appl. Chem. 71 (1999) 899.[5] D. Templeton, F. Ariese, R. Cornelis, H.P. van Leeuwen, L.G.

Danielsson, Pure Appl. Chem., in press.[6] G.K. Zoorob, J.W. McKiernan, J.A. Caruso, Mikrochim. Acta

128 (1998) 145.[7] R. Lobinski, Appl. Spectrosc. 51 (1997) 260A.[8] Ph. Quevauvillier, Method Performance Studies for Speciation

Analysis, Royal Society of Chemistry, Cambridge, UK, 1998.[9] J.C. Van Loon, Anal. Chem. 51 (1979) 1139A.

[10] J. Edmonds, M. Morita, Pure Appl. Chem. 64 (1992) 575.[11] L.A. Ellis, D.J. Roberts, J. Chromatogr. 774 (1997) 3.[12] R.M. Barnes, Fresenius J. Anal. Chem. 361 (1998) 246.[13] C. Casiot, V. Vacchina, H. Chassaigne, J. Szpunar, M.

Potin-Gautier, R. Lobinski, Anal. Commun. 36 (1999) 77.[14] E.H. Larsen, Spectrochim. Acta 53B (1998) 253.[15] S.J. Lippard, J.M. Berg, Principles of Bioinorganic Chemistry,

University Science Books, Mill Valey, CA, 1994.

[16] W. Kaim, B. Schwederski, Bioinorganic Chemistry: InorganicElements in the Chemistry of Life, Wiley, Chichester, 1994.

[17] A. Makarov, J. Szpunar, Analusis 26 (1998) M44.[18] C. Casiot, J. Szpunar, R. Lobinski, M. Potin-Gautier, J. Anal.

At. Spectrom. 14 (1999) 645.[19] M.S. Alaejos, C.D. Romero, Chem. Rev. 95 (1995) 227.[20] L.C. Clark, G.F. Combs Jr., B.W. Turnbull, E.H. Slate, D.K.

Chalker, J. Chow, L.S. Davis, R.A. Glover, G.F. Graham,E.G. Gross, A. Krongrad, J.L. Lesher Jr., H.K. Park, B.B.Sanders Jr., C.L. Smith, J.R. Taylor, J. Am. Med. Assoc. 276(1996) 1957.

[21] S.M. Bird, P.C. Uden, J.F. Tyson, E. Block, E. Denoyer, J.Anal. At. Spectrom. 12 (1997) 785.

[22] S.M. Bird, H.-h. Ge, P.C. Uden, J.F. Tyson, E. Block, E.Denoyer, J. Chromatogr. 789 (1997) 349.

[23] J. Zheng, W. Gössler, W. Kosmus, Trace Elem. Electrol. 15(1998) 70.

[24] E.O. Kajander, R.L. Pajula, R.J. Harvima, T.O. Eloranta,Anal. Biochem. 179 (1989) 396.

[25] J.F. Riodan, B.L. Valee (Eds.), Metallobiochemistry PartB. Metallothionein and Related Molecules, Methods inEnzymology, vol. 205, Academic Press, New York, 1991.

[26] M.J. Stillman, C.F. Shaw, K.T. Suzuki (Eds.), Metallo-thioneins Synthesis, Structure and Properties of Metallo-thioneins, Phytochelatins and Metalthiolate Complexes, VCH,New York, 1992.

[27] M.J. Stillman, Coord. Chem. Rev. 144 (1995) 461.[28] R. Lobinski, H. Chassaigne, J. Szpunar, Talanta 46 (1998)

271.[29] IUPAC, J. Biol. Chem. 252 (1977) 5939.[30] H. Chassaigne, R. Lobinski, Fresenius J. Anal. Chem. 361

(1998) 267.[31] G. O’Connor, L. Ebdon, E.H. Evans, H. Ding, L.K. Olson,

J.A. Caruso, J. Anal. At. Spectrom. 11 (1996) 1151.[32] H. Chassaigne, R. Lobinski, Anal. Chem. 70 (1998) 2536.[33] H. Chassaigne, R. Lobinski, J. Chromatogr. 829 (1998) 127.[34] X. Guo, H.M. Chan, R. Guevremont, K.W.M. Siu, Rapid

Commun. Mass Spectrom. 13 (1999) 500.[35] W.E. Rauser, Plant Physiol. 109 (1996) 1141.[36] M.H. Zenk, Gene 179 (1996) 21.[37] I. Leopold, D. Guenther, Fresenius J. Anal. Chem. 359 (1997)

364.[38] H. Chassaigne, V. Vacchina, M. Oven, M. Zenk, R. Lobinski

Analyst, in the press.[39] J.R. Yates III, A.L. McCormack, A.J. Link, D. Schieltz, J.

Eng, L. Hays, Analyst 121 (1996) 65R.

biochemical speciation analysis by hyphenated techniques

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