Chapter 19

CHAPTER 19 HYPHENATED TECHNIQUES IN ENVIRONMENTAL

SPECIATION ANALYSIS

Brice Bouyssiere, Ryszard Lobinski and Joanna Szpunar UMR 5034 CNRS, Hélioparc, 2, av. Pr. Angot, 64 000 Pau, France

ABSTRACT The recognition of the fact that 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 a rapid development of an area of analytical chemistry referred to as speciation analysis [1]. A fundamental tool used for speciation studies is the combination of a chromatographic separation technique, that ensures that the analyte compound leaves the column unaccompanied by other species of the analyte element, with atomic spectrometry, permitting a sensitive and specific detection of the target element.

Recent advances in the application of these hyphenated (coupled) techniques for species-selective determination of :

(i) volatile organo-metallic (Sn, Hg, Pb) anthropogenic contaminants

and (ii) non-volatile organometalloid (As, Se) compounds and heavy

metal complexes in environmental matrices are discussed. . Particular attention is given to the limitations related to chromatographic signal identification due to unavailability of standards for many species since a large number of the naturally synthesized compounds have not yet been identified and characterized. The potential of molecular mass spectrometry for this purpose is demonstrated.

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1 INTRODUCTION The recognition of the fact that, in environmental chemistry, occupational health, nutrition and medicine, 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 a rapid development of an area of analytical chemistry referred to as speciation analysis[1]. IUPAC defines a chemical species as a specific and unique molecular, electronic, or nuclear structure of an element[1]. Speciation of an individual element refers to its occurrence in or distribution among different species. Speciation analysis is the analytical activity of identifying and quantifying one or more chemical species of an element present in a sample[1]. The combination of a chromatographic separation technique, that ensures that the analyte compound leaves the column unaccompanied by other species of the analyte element, with atomic spectrometry, permitting a sensitive and specific detection of the target element, has become a fundamental tool for speciation analysis, as discussed in many review [2-7].

In the original concept[3], speciation analysis targeted well-defined analytes, usually anthropogenic organometallic compounds, such as alkyllead, butyl- and phenyltin compounds, and simple organoarsenic and organoselenium species, and products of their environmental degradation. Calibration standards were 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 efficiency and the absence of the condensed mobile phase, that enabled a sensitive (down to the femtogram levels) element-specific detection by atomic spectroscopy [8, 9].

Metalloids, such as arsenic and selenium, are known to be metabolised by living organisms in a way that leads to the formation of a covalent bond between the heteroatom and the carbon incorporated in a larger structure (e.g. arsenosugars, selenoproteins). Microorganisms and plants have developed a number of internal mechanisms to control the homeostasis of essential elements and to cope with the stress induced by toxic elements [10]. Some plants, referred to as hyperaccumulators, have evolved particularly efficient metal homeostasis mechanisms that allow them to live and reproduce in metal-rich environments. The resistance mechanisms include high turnover of organic acids (e.g. phytate, malate, citrate, oxalate) or induction and activation of antioxidant enzymes [10]. A well known mechanism of enhancing heavy metal accumulation and tolerance is the expression of metal-binding proteins or peptides in plants [11]. The complexation of metals leads to a number of relatively poorly characterized metal complexes. The understanding of mechanisms controlling the detoxification is possible only by the availability of analytical data on the species formed. The resulting compounds are difficult to be converted into volatile species that prevents the use of GC for their separation. Moreover, standards for most of these species are unavailable since many of the naturally synthesized compounds have not been identified and characterized yet. The analytical challenges spur therefore the use of separation techniques in the liquid phase, such as HPLC and CZE with ICP MS detection in combination with the use of molecule specific techniques, notably electrospray tandem mass spectrometry (ES MS/MS).

This paper reviews recent advances in the application of the hyphenated (coupled) techniques for species-selective determination of : (i) volatile organo-metallic (Sn, Hg, Pb) anthropogenic contaminants and (ii) non-volatile organometalloid (As, Se) compounds and heavy metal complexes in

environmental matrices.

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Particular attention is given to the limitations related to chromatographic signal identification due to unavailability of standards for many species since a large number of the naturally synthesized compounds have not been identified and characterized yet. The potential of molecular mass spectrometry for this purpose is demonstrated. 2 ORGANOMETALL(OID) SPECIES IN THE ENVIRONMENT The species of interest, containing a covalent carbon-metal(loid) bond, in environmental speciation analysis can be divided into the following classes: (i) products of environmental methylation of mercury, selenium, arsenic, tin,

bismuth, or carbonylation of molybdenum and tungsten (Mo(CO)6, W(CO)6), (ii) organometallic anthropogenic contaminants and products of their degradation or

environmental transformation. This group includes tetraalkylated lead (EtxMeyPb, x+y = 4), an antiknock additive to gasoline, that is degraded to trialkyl or dialkyl species, ingredients of antifouling paints, such as butyl-, octyl and phenyl tin species, released into the aquatic environment, and products of their degradation or biomethylation,

(iii) products of the metabolism of arsenic by marine biota leading to the formation of the carbon-arsenic bond, as e.g. in arsenobetaine or arsenosugars,

(iv) selenoaminoacids, -peptides and proteins, biosynthesized by bacteria, fungi and plants

(v) metal-binding peptides which are enzymatically synthetized in living organisms exposed to heavy metal (Cd, Cu,…) stress.

3 HYPHENATED TECHNIQUES IN SPECIATION ANALYSIS A suitable analytical technique for speciation analysis should address three issues: (i) selectivity of the separation technique allowing the target analyte species to

arrive at the detector well separated from potential matrix interferents and from each other,

(ii) sensitivity of the element or molecular selective detection technique since the already low concentrations of trace elements in environmental samples are usually distributed among several species,

(iii) species identification. Tetention time matching usually employed requires the availability of standards. When standards are non available, the use of a molecule-specific detection technique is mandatory.

The above challenges can be addressed by a hyphenated technique of which the

choice available is schematically shown in Figure 1. In the most frequent case a separation technique: chromatography (gas or liquid), electrochromatography or gel electrophoresis is combined with ICP MS. The coupling is realized directly (for GC), via a nebulizer (for column liquid separation techniques) or by laser ablation (for planar techniques).

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The separation component of the coupled system becomes of particular concern when the targeted species have similar physicochemical properties. Gas chromatography should be chosen wherever possible because of the high separation efficiency and the very low achievable detection limits because of the absence of the condensed mobile phase. For non-volatile species column liquid phase separation techniques, such as HPLC and CE, are the usual choice because of the ease of on-line coupling and the variety of separation mechanisms and mobile phases available allowing the preservation of the species identity. The two-dimensional gel electrophoresis is indispensable in seleno- and phosphoroproteomics because of its impressive peak capacity.

For element-specific detection in gas chromatography, a number of dedicated spectrometric detection techniques can be used, e.g. quartz furnace atomic absorption or atomic fluorescence for mercury, microwave induced plasma atomic emission for lead or tin, but it is ICP MS that has been extablishing its position as the versatile detector of choice. ICP MS is virtually the only technique capable of coping, in on-line mode, with the trace element concentrations in LC and CE effluents. The femtogram level absolute detection limits may turn out to be insufficient if an element present at the ng/mL level is split into a number of species, or when the actual sample amount analysed is limited to several nanolitres as in the case of CE.

Electrospray MSElectrospray MS

MALDI MSMALDI MS

AffinityAffinity

HPLCHPLC

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chromatography

GaschromatographyGas

chromatography

PackedcolumnPacked

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column

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Reversed-phaseReversed-phase

CZECZE

MEKCMEKC

CECCEC

Isotope intensitymeasurement(Q, TOF, SF)

Isotope intensitymeasurement(Q, TOF, SF)

Isotope ratio measurement

(TOF, MC)Isotope ratio

measurement(TOF, MC)

Isotopedilutionanalysis

Isotopedilutionanalysis

Tracer studiesTracer

studies

FT ICR FT ICR

CID MS(QqQ, QqTOF)CID MS

(QqQ, QqTOF)

Ion Trap MSIon Trap MS

postsourcedecaypostsource

decay

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ICP MS detectionICP MS detection

IdentificationIdentification

SeparationSeparation

Electrospray MSElectrospray MS

MALDI MSMALDI MS

AffinityAffinity

HPLCHPLC

ElectrochromatographyElectro

chromatography

GaschromatographyGas

chromatography

PackedcolumnPacked

column

MulticapillarycolumnMulticapillary

column

Size-exclusionSize-exclusion

Ion-exchangeIon-exchange

Reversed-phaseReversed-phase

CZECZE

MEKCMEKC

CECCEC

Isotope intensitymeasurement(Q, TOF, SF)

Isotope intensitymeasurement(Q, TOF, SF)

Isotope ratio measurement

(TOF, MC)Isotope ratio

measurement(TOF, MC)

Isotopedilutionanalysis

Isotopedilutionanalysis

Tracer studiesTracer

studies

FT ICR FT ICR

CID MS(QqQ, QqTOF)CID MS

(QqQ, QqTOF)

Ion Trap MSIon Trap MS

postsourcedecaypostsource

decay

CapillarycolumnCapillary

column

ICP MS detectionICP MS detection

IdentificationIdentification

SeparationSeparation

Figure 1. The most popular hyphenated techniques available for trace speciation analysis.

The isotope specificity of ICP MS offers a still underexploited potential for tracer studies and for improved accuracy in quantification via the use of isotope dilution techniques.

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The third important component of an analytical strategy is identification and characterization of metallospecies, either newly discovered, or those for which standards are unavailable. This can be achieved by electrospray MS or MALDI MS for column or planar separation techniques, respectively. The use of TOF MS is recommended; the 5-10 ppm accuracy of the Mr measurement allows the determination of the empiric formula for metallospecies with Mr < 500. Structural information can be acquired by collision induced dissociation (CID) of an ion selected by a quadrupole mass filter followed by a product ion scan using a quadrupole or a TOF mass analyser. Despite recent considerable advantages in instrumentation, an enzymic digestion of a selenopolypeptide prior to aminoacid sequencing by ES MS/MS, is necessary to complete the characterization. 4 ELEMENT SELECTIVE DETECTION IN GAS CHROMATOGRAPHY The practical applications to volatile organometallic species are dominated by the three techniques: GC - MIP AED, GC - ICP MS and GC - EI MS. The only exception is the determination of methylmercury in the environment where the position of GC-AAS and GC-AFS is still remarkably strong. A similar statement applies, however to a lesser degree, to organotin analysis that can still be successfully performed by GC - FPD, especially when the improved pulsed flame photometric detector is used. MIP AES will be prefered during the analysis of S, F and Cl of which the ionization in an ICP is extremely difficult. The improved ion optics and hence the sensitivity of the ICP MS spectrometers compensates for this lack, especially that the use of collision cell can improve the selectivity.

The combination of capillary GC with ICP MS has become an ideal methodology for speciation analysis for organometallic compounds in complex environmental and industrial samples because of the high resolving power of GC and the sensitivity and specificity of ICP MS. Indeed, the features of ICP MS such as low detection limits reaching the one femtogram (1 fg) level, high matrix tolerance allowing the direct analysis of complex samples, such as e.g. gas condensates, or the capabibility of the measurement of isotope ratios enabling accurate quantification by isotope dilution position ICP MS at the lead of the GC element specific detectors.

The ICP quadrupole MS is undergoing a constant improvement leading to a wider availability of more sensitive, less interference prone, smaller in size and cheaper instruments which favours their use as chromatographic detectors. The introduction of ICP time-of-flight MS increased the speed of data acquisition allowing multiisotope measurement of millisecond-wide chromatographic peaks and improving precision of isotope ratios determination[12-16]. An even better precision was recently reported for magnetic sector multicollector instruments used as on-line GC detectors [17, 18]. These instrumental developments go in parallel with the miniaturization of GC hardware allowing the time-resolved introduction of gaseous analytes into an ICP, e.g. based on microcolumn multicapillary GC, and sample preparation methods including microwave-assisted, solid phase microextraction or purge and capillary trap automated sample introduction systems [19].

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The basic requirement for an interface is that the analytes should be maintained in the gaseous form during transport from the GC column to the ICP, in a way that any condensation is prevented. This can be achieved either by an efficient heating of the transferline avoiding the cold spots, or by using an aerosol carrier. This results in two basic types of designs of the GC - ICP MS interface. (i) based on direct connection of transferline to torch [20]. (the spray chamber is

removed and the transfer line inserted part of the way up the central channel of the torch);

(ii) based on mixing the GC effluent with the aqueous aerosol in the spray chamber prior to introduction into the plasma detectors [17, 18]. Regardless of the type of the interface an addition of oxygen to the plasma gas is

essential to prevent carbon deposition (and sometimes metal entrapment) and to reduce the solvent peak. The interface designs were reviewed in detail[9].

GC - ICP MS interfaces are commercially available having proven the recognition of the maturity of this coupling by analytical instrumentation industry 4.1 Advances in Gas Chromatography prior to Element Selective Detection Packed column GC used in the early studies on GC - ICP MS coupling [21, 22] has practically given way to capillary GC; the coupling of the latter to ICP MS was first described by Kim et al. [23, 24]. Packed columns can, by design, handle high flow rates and large sample sizes, but the efficiency and resolution properties are compromised because of the high dispersion of the analytes on the column. The large column volume negatively affects the sensitivity in the peak height mode and the detection limits. The packing itself may be chemically active toward many organometallic species, which makes silanization necessary and worsens the reliability of results. It should be noted, however, that still a considerable number of works, especially those using hydride generation purge and trap are carried out with packed column chromatography because of easier handling of highly volatile species at temperatures below -100 ° C [25, 26].

Capillary GC offers improved resolving power over packed column GC - ICP MS which is of importance for the separation of complex mixtures of organometallic compounds found in many environmental samples. Capillary GC allows one to cope with the co-elution of the solvent and the volatile compounds, such as Me4Sn or Me2Hg, and thus to avoid or to minimize the plasma quenching. The reduced sample size and the high dilution factor with the detector's makeup gas necessary to match the spectrometer's optimum flow rate result in a loss of sensitivity.

Recently, a number of papers have appeared on rapid (flash) GC employing columns that consist of a bundle of 900 - 2000 capillaries of a small (20-40 µm) internal diameter, referred to as multicapillary columns (for a review see [27]). Such a bundle of capillaries allows the elimination of the deficiencies associated with the use of capillary and packed columns while the advantages of both are preserved. Multicapillary GC features high flow rates which minimize the dilution factor and facilitate the transport of the analytes to the plasma. The coupling between MC GC and ICP MS using a non-heated interface offered 0.08 pg detection limits for Hg speciation [28].

An interesting feature is the use of multicapillary microcolumns for sample introduction into an ICP. Rodriguez et al. [20] showed isothermal separations of organometallic species using a 50-mm long microcolumn which opens the way to the miniaturisation of GC sample introduction units, possibly making the classical GC oven redundant.

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4.2 Element Seletive Detection in Gas Chromatography Flame AAS initially used was quickly abandoned because of insufficient sensitivity preventing applications to real-world samples and an electrothermally heated silica tube is usually used as the atomization cell [29, 30].

An AFS detector coupled with a gas chromatograph is a commercially available hyphenated system allowing speciation of mercury [31]. GC - AFS is a convenient method for mercury speciation in environmental matrices but the risk of artefacts due to the presence of hydrocarbons prevents this technique’s use in successful applications to more complex matrices.

Despite problems with complete elimination of background and hydrocarbon interferences, FPD enjoys a strong position as a tin selective detector. A 600-610 nm bandpass filter is usually applied to avoid the sulfur interference. The need for the chemical elimination of sulfur was frequently evoked during the analysis of sediments in order to avoid the formation of artefacts [32, 33]. An advance in flame photometric detection is the pulsed flame photometric detector (PFPD), a 10-fold gain in sensitivity for organotin detection was claimed [34, 35].

Mass spectrometry of molecular ions, which is a common detection technique in GC of organic compounds, is relatively seldom used in speciation analysis. For quantitative analysis the widest popularity was enjoyed by electron impact mass spectrometres operated in the single ion monitoring mode for which detection limits are two orders of magnitude lower than in the full scan mode for structure elucidation. For most organometallic compounds detection limits at the low picogram level can be achieved in the single ion monitoring mode.

Plasma detectors compare favourably with spectrometers listed above. The use of different plasmas for element-specific detection in GC effluents was critically reviewed [8]. The practical significance of these studies in terms of applications is almost non existent with the exception of the microwave induced plasma.

The coupling of GC - MIP AED has been extremely popular in speciation analysis of anthropogenic environmental contaminants and products of their degradation. This was due to its versatility and the detection limits in the sub-picogram detection limits that could be matched only by ICP MS[8]. Another factor contributing considerably to the popularity of GC - MIP AED has been the commercial availability of an instrument.

GC - MIP AED offers sufficiently attractive figures of merit to be applied on a routine basis to speciation of organotin and organolead compounds in the environment and methylmercury in biological tissues. It is being gradually replaced by GC - ICP MS whose lower detection limits allow a simpler sample preparation procedure, work with more dilute extracts, and especially a sensitive speciation analysis of complex matrices. The position of ICP MS has recently become stronger owing to the availability of the commercial interface and this detection technique is discussed in detail in the subsequent section. 4.3 ICP MS Detection in Gas Chromatography Quadrupole mass analysers have predominantly been used, their sensitivity has improved by a factor of 10 during the past decade. Tao et al.[36] reported a fabulous instrumental detection limit of 0.7 fg by operating the shield torch at normal plasma conditions using the HP 4500 instrument. Of other types of analysers, TOF MS has been extensively studied as a GC detector during the last few years [14-16, 37]. Applications of sector-field analysers [17, 18, 38], also with multicollectors [17, 18],are slowly appearing.

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4.3.1 GC - ICP Time-of-Flight MS The measurement of a time-dependent, transient signal by a sequential scanning using a quadrupole or a sector-field (single collector) mass spectrometer results in two major types of difficulties. The first one is the limited number of isotope intensity measurements that can be carried out within the time-span of a chromatographic (especially from capillary or multicapillary GC) peak. The other one is the quantifi-cation error known as spectral skew [39],that arises during the measurement of adjacent mass-spectral peaks at different times along a transient signal. Alleviating these difficulties requires increasing the number of measurement points per time unit and the simultaneous measurement of the isotopes of which the ratio is investigated.

The ability to produce complete mass spectra at a high frequency (typically > 20 000 s-1) makes TOF MS nearly ideal for the detection of transient signals produced by high speed chromatographic techniques. The simultaneous extraction of all m/z ions for mass analysis in TOF MS eliminates the quantification errors of spectral skew, reduces multiplicative noise and makes TOF MS a valuable tool for determining multiple transient isotopic ratios [14-16, 37]. Whereas some of GC - ICP TOF MS applications reported so far [14, 15, 37] could have readily been carried with a quadrupole mass analyzer, the emerging potential of ICP - TOF MS is evident in three areas: (i) as a diagnostic tool for tracer experiments (ii) for studying isotope fractionation reactions in the biovolatilization processes of

metals, (iii) for truly multielemental (above 3 elements) screening for volatile metal(loids) in

a sample. The practical problems to be addressed in GC - ICP TOF MS include:(i) the need for the removal of C+ ions originated from the solvent that would otherwise overload the detector. This is realised by use of the transverse rejection ion pulse option. Ions are deflected by means of a high voltage pulse applied perpendicularly at a time appropriate for the ion being rejected [15], (ii) the theoretically achievable number of acquired mass spectra per second is

impressive but the tremendous volume of data collected at this speed makes it necessary to be limited. Data acquisition speed of 200 individual spectra per second was found sufficient for data acquistion every 10 ms which allows the measurement of all but the fastest transient signals (peak widths <50 ms) [14],

(iii) the lower sensitivity in the monoelemental mode in comparison with the last generation of ICP quadrupole mass spectrometers. A minimum of appr. 500 pg of each species is necessary for the measurement of isotope ratios with a precision better than 0.5 % [16]. The limitations of the pulse counting system are clearly seen, with peak heights of more than 2000 counts reaching saturation (for an integration time of 100 ms) [16]. On the other hand, Heisterkamp et al. [15] reported a DL of 10-15 fg for alkyllead compounds, a value which is comparable with ICP Q MS. It should be emphasized that the loss of sensitivity in the monoelemental mode is compensated by the fact that the number of isotopes determined during one chromatographic run is no longer limited by peak definition (like in ICP MS employing a quadrupole analyser), because the number of data points per chromatographic peak is independent of the number of measured isotopes.

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4.3.2 GC - ICP MS using Sector Field Mass Analysers The hitherto works have been scarce [17, 18, 38]. Prohaska et al. used an ICP sector-field double-focussing mass spectrometer for the analysis of arsenic hydrides and organoarsenic compound in gaseous emissions from a microcosm, after their separation on a capillary column [40]. Neither instrumental detection limits were reported nor chromatograms were, however, shown. Sanz-Medel's group proposed the same technique for the detection of 32S in GC analysis of volatile sulfur compounds in bad breath and reported, when a guard electrode was used under cold plasma conditions, absolute DLs in the low nanogram range[38].

The other two reports concerned the precise measurement of isotopic ratios by GC - ICP MS with multicollector detection factility [17, 18]. Four lead, 203Tl, 205Tl (for the correction of the mass bias) and 202Hg (for the correction of the 204Pb isobaric overlap) were monitored simultaneosly. A double-focussing instrument applied for this purpose allowed a detection limit of 1 pg for 207Pb (introduced as Et4Pb) which is poorer than values reported with other ICP mass spectrometers [17]. The minimum time resolution was limited by the Axiom software to 50 ms; 60 points could define a full peak width of 3 s [17]. Much lower detection limits were obtained by GC coupled to a single magnetic sector instrument equipped with a hexapole collision cell. A value of 2.9 fg was reported for the most abundant 208Pb isotope[18]; the values cited for the other isotopes were proportional to the isotopic abundance. 4.3.3 GC - ICP MS studies using stable isotopes The use of enriched isotopes with ICP MS detectors has been of benefit for the development of speciation methodology. The isotopic specificity of ICP MS opens the way to the use of stable isotopes or stable isotope enriched species for studies of transformations and of artefact formation during extraction and derivatization processes and to the wider implementation of the isotope dilution quantification. The latter had until recently been limited by the non-availability of organometallic species with the isotopically enriched element. However, standards for the isotopically enriched Me201Hg [41], MBT, DBT and TBT [42] have recently been synthesized and applications are being developed. The prerequisite of the use of stable dilution techniques is the precise and accurate measurement of the isotopic ratios. The to date applications to real-world samples have been exclusively carried out with ICP Q MS but precision and accuracy values for the measurement of isotope rations in standard compounds by ICP TOF MS [14] and by sector-field multicollector [17, 18] instruments has been reported.Tracer experiments can be performed with ICP Q MS but for the natural fractionation studies it may not be sufficient. 4.3.4. Isotope ratios measurements In GC- ICP MS the isotope ratio determinations are more precise if the intensities of the isotopes are integrated over the whole chromatographic peak instead of only measuring the isotope ratio at a single point of the peak [43]. A precision of 1% was reported for the Hg isotope ratios determined for MeEtHg eluted from a packed column by GC - ICP MS [44]. Heumann et al. reported a 0.5% precision for Se (derivatized as piazselenol) in GC Q ICP MS [43].

A tin isotope-ratio measurement accuracy of 0.28% and a precision of 2.88% was calculated for a 1-s wide GC peak of Me4Sn [14]. Haas et al. reported that a minimum of 0.5 ng of an organometallic species was necessary for the measurement of isotope ratios with a precision better than 0.5%, the best value (0.34%) was attained for Me2SnH2 [16]. ICP TOF MS should give an order of magnitude better precision than

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ICP Q MS in comparable conditions when natural gas samples were analyzed [16]. The precision of the determination of isotopic ratios is improved by the use of simultaneous multi-elemental ion extraction [15].

The precision of the isotope ratio measurements can be improved by using a sector field instrument with a multicollector facility. The precision values reported for the measurement of major Pb isotope ratio with a double-focusing instrument were better than 0.07% (for a 3-s transient signal) corresponding to a 0.35% accuracy [17]. When a single magnetic sector instrument (with a hexapole collision cell and multicollector detection) was used the precision was in the range 0.02 - 0.07% for ratios of high-abundance isotopes and injections of 5-50 pg [18]. After mass bias correction the accuracy was within 0.02-0.15% [18].

For accurate determinations by the isotope dilution technique the mass discrimination effect must be taken into account. Mass bias was about 0.5% per mass unit.17 The ways to measure and to correct for it included the sequential measurement of the isotope ration in the sample and the standard [45] or the addition of an internal standard, such as e.g. Cd [15, 16] or Tl [17, 18] and the simultaneous measurement of the 111Cd/ 113Cd or 203Tl/205Tl isotopic ratios, respectively. From a practical point the latter system requires the simultaneous delivery of the analyte and of the internal standard to the ICP MS which can be done only, via a spray-chamber interface in view of the nonvolatility of Cd and Tl species.

In order to enable an alternate measurement of the isotope ratio of the analyte element in a standard, a diffusion cell containing pure chemical of the element to be determined for calibration of the measured isotope ratios was proposed [45]. It consisted of a glass vial covered by a membrane which allowed diffusion of the volatile calibrant species into the flow cell. If the isotope ratios of the element in the calibration compound are known, the measured isotope ratio of the separated species in the sample can be corrected [45]. 4.3.5 Speciated isotope dilution analysis Isotope dilution (ID) MS is a method of proven high accuracy. The sources of systematic errors are well understood and can be controlled which makes ID MS accepted as a definitive method of analysis. Fundamentals of ID GC - ICP MS for species-specific analysis were extensively discussed by Gallus and Heuman [45]. They were illustrated by the determination of Se(IV) in water after conversion of the analyte species into piazselenol [45].

In ID GC - ICP MS the sample is spiked with the species to be determined in which one of the isotopes of the metal or metalloid was enriched. After equilibration of the spike, the sample preparation procedure, GC-ICP MS is run and the isotopic ratio of the metal(loid) in the species of interest is measured. The analysis principle is identical as in classical ID ICP MS, however, some fundamental differences occur.

The speciated isotope dilution analysis is only possible for element species well defined in their structure and composition. The species must not undergo interconversion and isotope exchange prior to separation. The equilibration of the spike and analyte, attainable in classical ID thermal ionization MS by multiple sequential dissolution and evaporation to dryness cycles, cannot be guaranteed to be achieved for speciated ID analysis in solid samples. Consequently, the prerequisite of the ID method: that the spike is added in the identical form as the analyte is extremely difficult, not to say impossible, to attain. Nevertheless, some advantages, such as the inherent corrections for the loss of analyte during sample preparation, for the incomplete derivatization yield, and for the intensity suppression/enhancement in the plasma are

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evident. In particular, ID quantification seems to be attractive in speciation analysis of complex matrices (e.g. gas condensates) when the different organic consituents of the sample modify continuously the conditions in the plasma and thus the sensitivity [46].

Isotopically enriched species should represent the ultimate means for specific accurate and precise instrumental calibration. Not only they are useful for routine determination by speeding analysis, but they also assist in the testing and diagnostics of new analytical methods and techniques. To date, the application examples of speciated ID GC - ICP MS have been relatively scarce. The determination of dibutyltin in sediment was carried out by ID analysis using an 118Sn-enriched spike. No recovery corrections for aqueoeus ethylation or extraction into hexane were necessary and no reaarrangement reactions were evident from the isotope ratios [47]. A mixed spike containing 119Sn enriched mono-, di- and tributyltin was prepared by direct butylation of 119Sn metal and characterized by reversed isotope dilution analysis by means of natural mono-, di- and tributyltin standards. The spike characterized in this way was used for the simultaneous determination of the three butyltin compounds in sediment CRMs [42]. Isotopically labelled Me2Hg, MeHgCl and HgCl2 species were prepared and used for the determination of the relevant species in gas condensates with detection limits in the low pg range [46]. 4.4 Advances in Sample Preparation for GC-based Hyphenated Techniques In speciation analysis sample preparation is often troublesome and developments in this area are attracting considerable attention. The increased use of microwave-assisted extraction techniques in speciation analysis has been also reflected with regard to GC -ICP MS [19, 20, 28]. The most important recent advances in sample preparation included the introduction of NaBPr4 for the derivatization of organometallic species [48], and the use of headspace solid-phase microextraction (SPME) [49-54], stir bar sorptive extraction [55] and purge and capillary trapping for analyte recovery and preconcentration [56, 57]. 4.4.1 Derivatization Techniques Although some authors still use the classical DDTC extraction in the presence of EDTA followed by butylation for organolead speciation analysis [58], the position of tetraalkylborates allowing the derivatization in the aqueous phase, such as NaBEt4 for organomercury and organotin speciation analysis and the newly introduced NaBPr4 [48] for organolead is well established. Synthesis of NaBPr4 was described in detail [48]. The possibility of the simultaneous determination of Sn, Hg and Pb following the propylation was demonstrated [48]. Artefact formation with hydride generation of antimony was discussed [59].

Two careful comparison studies are worth-noting. In one of them three derivatization approaches: anhydrous butylation using a Grignard reagent, aqueous butylation by means of NaBEt4 and aqueous propylation with NaBPr4 were compared for mercury speciation.88 The absence of transmethylation during the sample preparation was checked using a 97% enriched 202Hg inorganic standard [60]. In another study two different derivatisation approaches: esterification of the carboxylic selenomethionine group using 2-propanol followed by the acylation of the amino group with trifluoroacid anhydride and the simultaneous esterification and acylation with ethyl chloroformate-ethanol were compared for the determination of selenomethionine by GC - ICP MS [61]. The structure of the derivatised aminoacid was confirmed in parallel by GC – MS [61].

Derivatization techniques for GC were reviewed [62].

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4.4.2 Purge and Trap using Capillary Cryofocussing A semi-automated compact interface for time-resolved introduction of gaseous analytes from aqueous solutions into an ICP MS without the need for a full-size GC-oven was described [56]. The working principle was based on purging the gaseous analytes with an inert gas, drying the gas stream using a 30-cm tubular Nafion membrane and trapping the compounds in a thick film-coated capillary tube followed by their isothermal separation on a multicapillary column. Recoveries were reported to be quantitative up to a volume of 50 mL [56, 57, 63]. 4.4.3 Solid-Phase Micro-Extraction (SPME) Solid-phase micro-extraction (SPME) is a preconcentration technique based on the sorption of analytes present in a liquid phase or, more often, in a headspace gaseous phase, on a microfibre coated with a chromatographic sorbent and incorporated in a microsyringe. The analytes sorbed in the coating is transferred to a GC injector for thermal desorption. SPME is an emerging analytical tool for elemental speciation in environmental and biological samples [54]. This solvent-free technique offers numerous advantages such as simplicity, the use of a small amount of liquid phase, low cost and the compatibility with an on-line analytical procedures.

SPME is based on an equilibrium beetween the analyte concentrations in the headspace and in the solid phase fibre coating. Low extraction efficiencies are hence sufficient for quantification but the amount of the analyte available may be very small. Hence, the interest for the high sensitivity of GC - ICP MS to be combined with SPME.

The first work SPME - GC - ICP MS concerned speciation of organomercury, -lead and -tin compounds ethylated in-situ with NaBEt4 and sorbed from the headspace on a poly(dimethylsiloxane)-coated fused silica fibre [49]. Headspace SPME using a 100 µm polydimethylsiloxane fiber at no equilibrium conditions was optimized as an extraction/preconcentration method for triphenyltin residues in tetramethylammonium hydroxide (TMAH) and KOH-EtOH extracts of potato and mussel samples [51]. Tricyclohexyltin was used as an internal standard. Derivatization was carried out with NaBEt4 for 10-20 min [51]. Direct SPME (from the aqueous phase) was studied but the sensitivity was an order of magnitude lower. A detection limit of 2 pg l-1 was reported for an aqueous standard but a value of 125 pg l-1 was given for the sample extract correxponding to a DL in the low ng/g range (dry weight) [51]. Slightly lower detection limits (0.6 - 20 pg l-1) were reported in another work [64].

A direct coupling of SPME with ICP MS was described for specied-specific determination of methylmercury [53]. A fiber was inserted into a splitless-type GC injector which was placed directly at the base of the torch. Headspace was slightly less sensitive than immersion sampling but offered a larger linearity range. Immersion SPME was severley influenced by the matrix that lead to a 70-fold decrease in sensitivity [53].

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5 LIQUID CHROMATOGRAPHY WITH ICP MS DETECTION Many element species of interest in environmental speciation analysis are non volatile and cannot be converted into such by means of derivatization. They include virtually all the coordination complexes of trace metals but also many truly organometallic (containing a covalently bound metal or metalloid) compounds. For all these species HPLC is the principal separation technique prior to element selective detection.

The various possibilities of on-line coupling a separation technique with an element (species) specific detector for species-selective analysis of metallocompounds of biological origin include different modes of HPLC or electrophoresis in terms of separation, and atomic spectrometry (or molecular MS) in terms of detection. The presence of a metal bound to the biomacromolecule in a sample is considered to be the prerequisite of using an element-specific detector.

The choice of the hyphenated setup depends primarily on the research objective. The separation component of the coupled system becomes of particular concern when a high degree of species-specificity is required. It may even be necessary to combine two or more separation techniques in series to assure that a unique species arrives at a certain time at the detector. The choice of the detector component becomes crucial when the amount of analyte species is very small and a high sensitivity is necessary - the most popular choice is ICP MS. An important problem is often the interface between chromatography and spectrometry as the separation conditions may be not compatible in terms of flow rate and mobile phase composition with those required by the detector.

In contrast to the speciation analysis [65] of the anthropogenic pollutants (realized mostly by GC-ICP MS) for which analytical standards are available, the majority of species of interest in biological environmental trace analysis have not yet been isolated sufficiently pure to be used as retention time standards. Therefore, it is becoming of paramount importance to employ in parallel a molecule (or moiety) specific detector to know the identity of the eluted species. Mass spectrometry: fast atom bombardment (FAB MS) [66-68], electrospray (ES MS) [69-72] or matrix assisted laser desorption ionization mass spectrometry (MALDI TOF MS) [73] have been the viable choices. 5.1 The coupling HPLC - ICP MS The principal HPLC separation mechanisms used in environmental 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 the high separation efficiency, the nanolitre sample requirement, and the absence of packing susceptible to interact with metals and to affect the complexation equilibria [74-76]. The combination of electrophoretic and electroosmotic flows provides the ability to separate a wide variety of positive, neutral and negative ions and compounds in one run. The complexity of the biological matrix may require the combination of two or more separation mechanisms in series to assure that a unique metal-species arrives at the detector at a given time.

The use of a quadrupole mass analyser in ICP MS detection is the most widespread. The latest generation of instruments offers sub-femtogram absolute detection levels for many elements. The isobaric overlaps are generally not a problem because of the on-line separation from the potential interferents, e.g. Cl (40Ar35Cl) in the case of 75As determination, but ghost peaks may appear. The application of a double focusing sector field instrument offers the higher resolution that may be required for the interference-free determination of sulfur or arsenic. An increase in resolution inevitably leads, however, to a dramatic decrease in sensitivity. It should also be noted that the

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sensitivity of the latest generation quadrupole instruments is only a factor of 2-3 lower than that of high resolution ICP MS operated in the low resolution mode. A good tradeoff between sensitivity, freedom from isobaric interferences and price is offered by ICP MS instruments equipped with a collision cell that have recently proliferated on the market [77].

The key to a succesful HPLC/CE - ICP MS coupling is the interface.In the simplest case the exit of an HPLC column (4.6-10 mm) is connected to a conventional pneumatic or crossflow nebulizer. The use of capillary or megabore (0.32 - 1.0 mm) HPLC systems that are becoming popular especially for reversed phase chromatography, requires the use of micronebulizers, either direct injection (DIN, DIHEN) or micronebulizers (e.g. Micromist) fitted with a small-volume nebulization chamber. The CE - ICP MS coupling is less straightforward. The problems due to the laminar flow generated by the nebulizer suction, loss of sensitivity because of the electroosmotic flow dilution by the makeup liquid and peak broadening in the spray chamber have been resolved in the commercially available interface based on a total-consumption self-aspirating micronebulizer fitted with a small-volume spray chamber [78, 79]. 5.2 Sample preparation Soluble extracts of tissues and cultured cells are prepared by homogenizing the sample in an appropriate buffer. Neutral buffers are normally used for the extraction since Zn starts dissociating from protein complexes at pH 5. Cd and Cu are removed at lower pH values. A 10 - 50 mM Tris-HCl buffer at pH 7.4 - 9 is the most common choice.

The filtration of the cytosol using a 0.45 µm or, better, a 0.22 µm filter before injection onto the chromatographic column is mandatory. A guard column should be used to protect the analytical column particularly from effects of lipids from animal tissues, that would otherwise degrade the separation. A number of bioanalytical techniques including ultracentrifugation, microdialysis, ammonium sulfate precipitation should often precede the chromatographic separations of metallocompounds.

The low yields of the aqueous leaching procedures for some species and samples promoted more aggressive leaching media to be used by some researchers. A trade-off is often necessary between the recovery from a solid matrix and the preservation of the original species.

Cellulose and complex water-insoluble pectic polysaccharides are the main matrix of the water insoluble residue after centrifugation of plant homogenates. The use of pectolytic enzymes is therefore necessary to solubilize the solid sample. Pectinolysis is known to degrade efficiently large pectic polysaccharides but some of them, e.g. rhamnogalacturonan-II are considered enzyme-resistant [80]. A mixture of commercial preparations: Rapidase LIQTM and Pectinex Ultra-SPLTM was reported for release of metal-complexes from the solid parts of edible plant, fruits and vegetables [80]. Extraction of selenium compounds from selenium-enriched yeast with a mixture containing a proteolytic enzyme led to recoveries of Se species above 85%, the majority as selenomethionine [81].

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5.3 Electrospray mass spectrometry for the species identification The access to structural information for the identification of known or novel compounds is a great challenge to speciation analysis, especially that the improving sensitivity of ICP MS instruments will inevitably increase the number of metal and metalloid species detected. Several recent reports have indicated exciting potential opportunities offered by electrospray mass spectrometry (ESI MS) for the precise determination of molecular weight and the structural characterization of molecules at trace levels in fairly complex matrices. This advantage leads to an increasing number of ESI MS reports gaining a complementary role to ICP MS for species identification following pre-fractionation using chromatographic methods with element selective detection. The use of ESI in this manner is, however, far more challenging, due in part to characteristically poorer signal-to-noise and signal-to-background ratio as well as more complicated gas-phase ion chemistry than typically encountered in an ICP. The various facets of electrospray mass spectrometry in speciation analysis have been reviewed [82, 83].

Molecules containing a carbon - metal (metalloid) bond usually produce readily singly protonated ions in the electrospray source that theoretically should allow the identification of the metallocompound on the basis of the molecular mass. However, in the direct infusion mode, the attribution of a signal at a given m/z ratio to an elemental species is a daunting and practically impossible task for monoisotopic elements, such as arsenic. However, if an element presents a characteristic isotopic pattern such as Se [84-90] or Sn [91, 92], its recognition in the mass spectrum of a sample solution is easier provided that the signal is not suppressed by the matrix.

A deeper insight into the species identity can be gained by the fragmentation of the protonated molecule ion (isolated at the level of the first mass filter) by collision induced dissociation (CID) followed by mass spectrometry of the product ions. The MS/MS mode allowed the identification of organoarsenic compounds in algal extracts purified by SE HPLC [93]. For species containing an element having more than one stable isotope such as Se, valuable information can be obtained by fragmenting the two protonated molecule ions containing the adjacent most abundant isotopes (78Se and 80Se). Fragments that contain selenium will still be separated by the distance of two units whereas fragments that do not will remain at the same m/z value thus facilitating the interpretation of the mass spectra [84].

Interpretation of mass spectra becomes easier when ES MS is used as a chromatographic [87, 89, 94, 95] or electrophoretic [96-98] detector. The source collision induced dissociation mode allows the use of ES MS as an element selective detector [99]. The elemental ES MS mode is free from many polyatomic interferences present in ICP MS. The concentration detection limits, are however 2-3 orders of magnitude higher than in the case of ICP MS.

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Migration time, min

x 107

5 10 15 20 25

Inten

sity,

cps

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(a) SEC-ICP MS

(b) CZE-ES MS

(c) ES MS

Figure 2: Analytical strategy allowing standardless identification of phytochelatins in plant extract: (a) size-exclusion HPLC-ICP MS for detection of heavy metal (cadmium) containing fraction, (b) capillary zone electrophoresis - ES MS for separation of individual species, (c) ES MS mass spectrum taken at the peak apex showing the molecular mass of the species

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0 100 200 300 400 500 600 700 800m/z

Sign

al int

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C)786

657554

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768697594

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γGluγGluγGlu

γGluγGlu

CysCys

CysCys βAla

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2

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(d) ES MS/MS

(γGlu-Cys)3 (βAla)

iso-Phytochelatin (3) - (βAla)

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iso-Phytochelatin (3) - (βAla)

(e) formula

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(γGlu-Cys)3 (βAla)

iso-Phytochelatin (3) - (βAla)

(γGlu-Cys)3 (βAla)

iso-Phytochelatin (3) - (βAla)

(e) formula

Figure 2: Analytical strategy allowing standardless identification of phytochelatins in

plant extract: (d) ES MS/MS mass spectrum of the signal shown above (c) allowing to resolve the structure of the compound, (e) formula of the identified species.

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5.4 Multidimensional analytical strategy for the identification of phytochelatins in plants The complexity of elemental speciation in biological samples often makes it necessary to couple several different separation mechanisms in order to isolate the compound of interest prior to its characterization by electrospray MS. This approach has been successfully used to the characterisation of phytochelatins in plants exposed to the heavy metal stress [100-103]. Phytochelatins (cadystins) are short metal-induced sulfhydryl-rich peptides possessing the general structure: (γ-GluCys)n-Gly with n = 2-11. They are synthesized from glutathione in plants and fungi exposed to metal ions: Cd2+, Cu2+, and Zn2+. Metals are chelated through coordination with the sulfhydryl group in cystein. Two principal structures of phytochelatins reported in the literatureγ: Cadystin A (γ-EC)3G and Cadystin B (γ-EC)2G, where the γEC - unit stands for -γ-Glu-Cys.

The individual compounds present in the soybean extract are isolated using a two dimensional scheme (Fig.2) including size-exclusion chromatography couple dto ICP-MS (Fig. 2A) followed by capillary zone electrophoresis (Fig. 2b). The isolated demetallated species are analyzed by electrospray MS (Fig. 2c). The identity confirmation or identification of an unknown compound can be achieved by CID MS (Fig. 2d). A series of phytochelatin isoforms could be identified without the need for authentic retention time standards. This example of standartless analysis in an environmental biota shows usefuless of tandem MS in environmental speciation studies. 6 CONCLUSIONS Gas chromatography with ICP MS detection has reached maturity as the analytical technique for speciation of organometallic species in a variety of matrices. It shows comparable figures of merit with that of GC - MIP AED for standard applications including speciation of organomercrury, organolead and organotin in the environment but offers a number of advantages in cases where extremely low sensitivity, multielemental screening, precise isotope ratios measurements or the analysis of complex matrices are requiered.

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