Speciation of cadmium in plant tissues by size-exclusion chromatography with ICP-MS Detection Ve�ronique Vacchina, Katarzyna Po�ec�† and Joanna Szpunar* CNRS EP132, He�lioparc, 2, av. Pr. Angot, 64053 Pau-Pyre�ne�es, France. E-mail: joanna.szpunar@univ-pau.fr Received 17th June 1999, Accepted 12th August 1999 A systematic approach based on the coupling of high resolution size-exclusion chromatography (SEC) with ICP-MS detection was developed for the speciation of Cd complexes with oligopeptides known to be biosynthesized by plants exposed to metal stress (phytochelatins, PCs).The separation conditions were optimized using a series of standards prepared by incubation of cadmium with purified (GluCys)2Gly (PC2), (GluCys)3Gly (PC3) and (GluCys)4Gly (PC4) ligands. The formation of artefact species was investigated and conditions allowing the reproducible chromatography and quantitative determination of Cd species in the range 0.01–15 mg ml-1 were established.The method developed was applied to investigate the speciation of Cd in cytosols of plant tissues and plant cell cultures. In addition to Cd complexes with PC2, PC3 and PC4 that eluted at the times corresponding to standards, two other Cd species, one excluded from the column and the other with an apparent molecular mass of 6800 Da, were observed in all the samples investigated. The latter compound, isolated and analysed by electrospray MS-MS, turned out to be a mixed Cd complex with diVerent PC ligands. The species eluted in the void volume could not be identified.The method was validated by comparing the results obtained by SEC-ICP-MS with those obtained by reversed-phase HPLC with post-column derivatization of phytochelatin. ICP-AES or ICP-MS12 were used to determine the metal Introduction concentration oV-line in the eluted fractions. A faster, more Aquatic and terrestrial plants are the primary entry points of reliable and more elegant analysis turned out to be possible essential micronutrients and toxic metals (such as cadmium) by the coupling of SEC with ICP-MS and this has become the into the food chain leading to animals and humans.The most widely used technique to detect stable metallodetermination of the total concentration of heavy metals is a compounds in plants.3,13 The applications included a multiroutine method to monitor the exposure of a plant to environ- elemental screening for elemental species in a particular matrix, mental metal pollution but there is increasing evidence that a such as tea leaves,14 fruit and vegetable extracts15 or plant more suitable approach to investigate the ecotoxicity of heavy extracts,16 or the investigation of a particular class of commetals, their pathways in the ecosystem and their metabolism pounds, e.g., metalloproteases of bacterial origin,17 polyby living organisms is the identification, characterization and saccharide complexes of lead in wine,18 boron complexes in determination of the particular metal species involved.1–6 plant extracts19,20 or platinum metabolites in grass.6 Plants have developed a number of internal mechanisms to Biochemical studies of metal speciation by SEC-ICP-MS control the homeostasis of essential elements and to cope with have suVered from a number of limitations and pitfalls.21–24 the stress induced by toxic elements.The most frequent case The most pertinent include (i) the non-availability of standards is the biosynthesis of a ligand, such as a phenolic compound, which means that the only information to be gained is an organic acid or oligo- or polypeptide, that would be able to approximate molecular mass of the analyte species estimated complex the excess of the toxic element into a compound on the basis of the elution volume of the complex, (ii) unconinnocuous to the organism.2,4,5 In particular, a class of oligope- trolled recovery of the metallo-complex because of its sorption tides having the general formula (EC)nG (n=2–11), called or dissociation following the interaction with the column phytochelatins (PCs), was found to play a crucial role in the packing, (iii) formation of artefacts on the column because of detoxification and homeostasis of heavy metals in plants.4,5 the exchange of the analyte metal ion or of the ligand with The complexation of metals occurs through the cysteine sulfur metal ions or ligands adsorbed earlier on the column and atom, leading to a number of relatively poorly characterized (iv) dependence of the complex composition on the chemical metal complexes.The understanding of mechanisms con- conditions of the mobile phase. The prerequisite for developing trolling the detoxification is possible only with the availability a valid approach to speciation analysis of metallo-complexes of analytical data on the species formed. by SEC-ICP-MS is therefore the synthesis of standards (that Various analytical approaches have been proposed to study are usually not commercially available) and a careful evalumetal speciation in plants.The classical approaches were based ation of the chromatographic separation conditions. This on the use of an ultra- and diafiltration technique7,8 and size- approach becomes indispensable when the complex is fairly exclusion chromatography (SEC)9,10 for the separation of the labile, which is often the case of metal complexes with oligoindividual metal species, whereas atomic absorption spec- and polypeptides.25 trometry,7–9 total reflection XRF,10 c-ray spectrometry11 and Whereas a lot of research has been devoted to the characterization of ligands bioinduced as a response of a plant to the metal stress,4,5 few data are available on the actual †On leave from the Department of Chemistry, Warsaw University of Technology, ul.Noakowskiego 3, 00-664 Warsaw, Poland. complexes formed by these ligands with heavy metals.J. Anal. At. Spectrom., 1999, 14, 1557–1566 1557Recently, SEC-ICP-MS allowed the detection of a 13 kDa where.26 The fractions with the individual PC (PC2, PC3 and PC4) peaks were heart-cut. The acetonitrile was rotavaporated compound containing Cd and Cu in which PC2 and desglycine-PC were detected by ESI-MS.3,16 Since the stan- and the fractions were lyophilized. The heart-cut fractions were repurified in the same way to produce compounds used dards were not available, the exact nature of this compound was not elucidated.as standards below. The objectives of this research were to investigate the Preparation of the PC–Cd complexes. A 1 ml volume of the behaviour of oligopeptide–Cd complexes in SEC at the trace solution (100 mg ml-1) of an individual PC in 30 mM and ultratrace levels, to evaluate the potential of SEC-ICP-MS TRIS–HCl buVer (pH 7.5) was mixed with an equimolar coupling for speciation of cadmium in plant extracts and to amount of a neutral solution of Cd2+.develop a method for the quantification of the individual Cd species found. Analytical procedures Experimental Sample preparation. Cells were vacuum filtered and washed. Plants roots were cut from the rest of the plant and washed Instrumentation (in both cases only with water). From here on, both cells and plants were treated in the same way. They were frozen in Chromatographic separations were performed using a Model liquid nitrogen to break the cell walls, ground with a mortar 1100 HPLC pump (Hewlett-Packard, Wilmington, DE, USA) and pestle and extracted with water or 10 mM TRIS–HCl as the sample delivery system.Injections were made using a buVer (pH 8). They were centrifuged (30 min, 10 000 g, 4°C), Model 7725 injection valve with a 100 ml injection loop filtered and lyophilized. (Rheodyne, Cotati, CA, USA). All the connections were made A sample of 10 mg of the freeze-dried material was dissolved of PEEK tubing (0.17 mm id).An ELAN 6000 ICP mass in 1 ml of 30 mM TRIS–HCl buVer (pH 7.5). The solution spectrometer (Perkin-Elmer SCIEX, Thornhill, ON, Canada) was ultracentrifuged for 30 min at 50 000 rpm at 4 °C. Prior was used as an element-specific detector in HPLC. The column to SEC-ICP-MS analysis, the supernatant was diluted 20-fold eluate was introduced into the ICP via a cross-flowor Silene cucubalus analysis, 10-fold for Agrostis tenuis analy- fitted in a Ryton spray chamber.For total analyses samples sis and not diluted for maize and Rauwolfia serpentina analysis. were fed by means of a Minipuls 3 peristaltic pump (Gilson, No dilution was used when SEC was used prior to ESI- Villiers-le-Bel, France) that also served for draining the spray MS/MS analysis. chamber. Chromatographic data were processed using Turbochrom4 software (Perkin-Elmer, Norwalk, CT, USA). SEC and ICP-MS conditions. Three types of SEC columns All signal quantifications were performed in the peak area (300×10 mm id) (Pharmacia Biotech, Uppsala, Sweden) were mode.ESI-MS/MS measurements were performed using a investigated, namely Superdex Peptide HR 10/30 designed for Perkin-Elmer SCIEX API 300 electrospray triple-quadrupole the separation of peptides (optimum separation range 100– mass spectrometer. A Hitachi (Tokyo, Japan) Model 7000 kDa), Superdex 75 HR 10/30 with an exclusion limit of CS120GX refrigerated ultracentrifuge was used for the separa- 100 kDa and an eVective separation range between 3 and tion of the supernatant after leaching of Cd species from plant 70 kDa and Superdex 200 with an exclusion limit of 1300 kDa tissues and cell cultures.and an optimum separation range of 10–600 kDa. A TSK PWXL guard column (40×3 mm id) (Tosoh, Tokyo, Japan) Reagents, solutions and materials was always used. The columns were calibrated (UV detection Analytical-reagent grade reagents purchased from Sigma- was used) with the following standards: glutathione (Mr 307), Aldrich (St.Quentin Fallavier, France) were used throughout PC2 (Mr 539), PC3 (Mr 771), rabbit liver metallothionein–Cd unless specified otherwise. Water (18 MV) prepared with a complex (Mr 6918), cytochrom c (Mr 12 384), bovine albumin Milli-Q system (Millipore, Bedford, MA, USA) was used (Mr 66 000) and thyroglobulin (Mr 660 000). throughout. The columns were conditioned between the injections with Cell cultures of the plants Silene cucubalus, Agrostis tenuis a 2mM b-mercaptoethanol solution in 30 mM TRIS buVer and Rauwolfia serpentina were investigated.They were grown (pH 7.5) for 30 min followed by removal of the chelating for 4 d in a 300 mM Cd2+ solution. Seeds of maize (Zea mays reagent by conditioning the column with the mobile phase for L.) were sown in sand followed by the addition of Hoagland another 30 min. Care should be taken to avoid traces of metals nutrient solution.When the plant was large enough (10 d after in the conditioning mobile phase solution. The column congermination), 300 mM Cd2 + solution was added. The maize dition should be controlled prior to injection of a sample by was harvested 1 week after Cd addition. injecting a 2 mM b-mercaptoethanol solution. Typical peak height values observed for Cd and Cu were lower than 200 Standards and 500 counts s-1, respectively. Preparation of metal-free phytochelatins.The synthesis of PC–metal complexes were eluted with 30 mM TRIS–HCl phytochelatins was carried out according to Grill et al.26 In buVer (pH 8.5) at a flow rate of 0.75 ml min-1. A 100 ml brief, 2000–10 000 pkat of PC synthase27 were incubated at aliquot of the sample solution was injected. The mobile phase 25 °C with 1 mM GSH and 0.8 mM Cd(NO3)2 in 1120 ml of was degassed ultrasonically. ICP-MS measurement conditions the buVer solution (pH 8.0). NaN3 (0.02%) was added to (nebulizer gas flow, rf power and lens voltage) were optimized retard bacterial growth.daily using a standard built-in software procedure. The iso- Proteins were precipitated from the resulting solution by topes monitored were 114Cd and 65Cu. The SEC-ICP-MS the addition of (NH4)2SO4 to 85% followed by centrifugation optimized instrumental conditions were optimized daily using at 8000 g for 30 min. PCs were precipitated from the super- the standard built-in optimization algorithm.Calibration was natant by the addition of 20 ml of 1 M Cd(NO3)2 solution. performed using standard graphs in the range 0.01–15 mg ml-1 The precipitated mixture of PCs was centrifuged and washed (as PC) established with the Cd–PC2–4 standards prepared as twice with water. The washed precipitate was stable for several described above. months when stored at -20 °C. The precipitated PCs were dissolved in 3.5M HCl and ESI-MS-MS analysis. ESI-MS/MS was applied to identify Cd species for which the elution volume in an SEC-ICP-MS separated by semi-preparative HPLC by elution with a concentration gradient of acetonitrile in water as described else- chromatogram could not be matched by one of the standards 1558 J.Anal. At. Spectrom., 1999, 14, 1557–1566prepared as described above. The fraction containing such a peak was collected and lyophilized. It was dissolved in 500 ml of water containing 0.1% of trifluoroacetic acid (TFA) (pH 2.3), filtered and desalted by reversed-phase chromatography by elution with 0.1% TFA in water for 5 min followed by gradient elution with 0.1% TFA in acetonitrile–water (50+50 v/v) for a further 40 min at 0.75 ml min-1.The eluate of the first 5 min was discarded. To the rest of the eluate 5 mM (dithiothreitol ) (DTT) was added, acetonitrile was removed by rotavaporation and the remaining solution was freeze-dried. The dried material obtained was dissolved in 200 ml of 0.06M acetic acid in 30% methanol and analysed by ESI-MS.ESI-mass spectra were acquired (10 scans) in the range Fig. 1 EVect of the excess of cadmium on the synthesis of PC–Cd 50–1100 u using a 10 ms dwell time and a 0.5 u step size. The complex. The determination was performed by SEC-ICP-MS. The orifice potential was 40 V, the ionspray voltage was 4100 V intensity corresponds to the area of the peak corresponding to a and the ion multiplier potential was 2400 V. The signals in the particular phytochelatin. 1, (GluCys)2Gly (PC2); 2, (GluCys)3Gly mass spectrum were identified by MS/MS using collision- (PC3); 3, (GluCys)4Gly (PC4). induced dissociation and the product ion scan mode. The collision gas was nitrogen and the collision energy was varied from 25 to 50 eV to obtain optimum fragmentation. Purity of the PC–Cd standards. The purity of the PC–Cd complexes obtained was monitored by SEC-ICP-MS. The chromatograms of the Cd complexes with the individual Results and discussion phytochelatins are shown in Fig. 2(a)–(c). Fig. 2(d) shows a chromatogram corresponding to the complexes formed by Preparation of standard solutions of Cd complexes with incubating cadmium with a mixture of apo-PCs prior to phytochelatins separation of the individual PCs by reversed-phase chromatography. Phytochelatins (oligopeptides with up to 11 amino acids) can be readily obtained in large amounts in the reaction of Chromatograms of the PC2 and PC3 standard solutions show that in each of the cases one major complex is formed polymerization of glutathione in the presence of an enzyme, phytochelatin synthase.26,27 The reaction produces a mixture that elutes as a well defined peak.Values of the molecular weight can be assigned to these peaks by calibrating the of oligopeptides that can be isolated by precipitation with Cd from the supernatant remaining after the precipitation of column with a series of oligopeptide standards. The molecular masses calculated in this way for the PC2–Cd and PC3–Cd larger proteins with ammonium sulfate.The precipitate (soluble in fairly concentrated HCl) can be desalted by reversed- complexes with cadmium (pH 8.5) are 2300±500 and 3400±500 Da, respectively. These results agree with an earlier phase chromatography under acidic conditions under which the mixture of PCs is recovered in the metal-free (apo) form.26 observation that phytochelatin peptides form complexes with Cd2+ of Mr 2500 and 3600.29 Individual PCs (PC2, PC3 and PC4) (apoforms) can be isolated by heart-cutting of the peaks in the reversed-phase In the case of the PC4–Cd complex, the chromatogram is more complex. In addition to the PC4–Cd signal with an chromatogram and characterized by ESI-MS/MS as described elsewhere.28 The species obtained are metal-free and cannot estimated molecular mass of 5100±500 (at pH 8.5) two other intense peaks are present.One of them corresponds to the be used as calibration standards for SEC-ICP-MS.The preparation of metallated standards requires the reconstitution of elution volume of the PC3–Cd complex. This may be explained by a PC3 impurity in the standard that is also indicated by an PC–Cd complexes under optimized reaction conditions. The metal-free PCs can apparently be reassociated with ESI mass spectrum (not shown) of the apo-PC4 standard solution. The intensity of this peak in the SEC-ICP-MS heavy metal ions simply by mixing a metal ion with an apophytochelatin.Particular care was taken to avoid oxidation of chromatogram is nevertheless higher than one would expect from the ESI mass spectrum. The other peak corresponds to the sulfhydryl groups during reconstitution.26 Having this in mind, we investigated two procedures for the preparation of a compound with an apparent molecular mass of 6800 u. The identity of this compound is unknown but it is likely to PC–Cd complexes. In the first, NaBH4 was added to the reaction mixture during reconstitution to ensure in situ reduc- correspond to a PC–Cd species of a diVerent stoichiometry.A trace of species at the same elution volume is also observed ing conditions according to Grill et al.26 The alternative was to deoxygenate the reaction solutions by sparging with helium, in the SEC-ICP-MS chromatogram of the PC3 standard [Fig. 2(b)]. which was expected to be suYcient to ensure a non-oxidizing environment. The chromatogram of a solution prepared by the incubation of a crude (not subjected to reversed-phase HPLC) mixture It was found that the SEC-ICP-MS chromatograms of the PC–Cd complexes obtained by the two procedures were ident- of apo-PCs with Cd2+ shown in Fig. 2(d) contains three baseline separated peaks with elution volumes corresponding ical. Therefore, the more straightforward procedure based on mixing an apo-PC solution with that of Cd2+ ions in TRIS to the PC2, PC3 and PC4 standards. Neither the small impurities observed in the PC3 chromatogram nor the intense signal buVer was used throughout.The excess of Cd to be added was determined by adding increasing amounts of Cd to the at Mr 6800 can be seen. The reason may be some degradation (e.g., oxidation of PCs during the reversed-phase chromato- solution of an apo-PC standard. The increase in the peak height as a function of increasing Cd concentration is shown graphic purification and freeze-drying procedures of the individual PCs).The mixture of PCs used to obtain the in Fig. 1. A PC-to-Cd ratio of 151 was applied for the reconstruction of PC–Cd complexes. Despite it being higher chromatogram in Fig. 2(d) was not subjected to any chromatographic or lyophilization steps (see Analytical procedures). than the stoichiometric ratio, it was chosen in view of the possible interactions of the complex with the column stationary phase. Note that the excess of Cd in the solution is anyway Stability of PC–Cd standard solutions with time.Fig. 3 shows the overlapped chromatograms of a standard solution freshly retained on the column. J. Anal. At. Spectrom., 1999, 14, 1557–1566 1559Fig. 3 Stability of standard solution of PC–Cd complexes with time. Chromatograms were obtained by SEC-ICP-MS. Solid line, freshly prepared solution of PC–Cd complexes; dashed line, solution 12 d old. Peaks: 1=unidentified; 2=PC4; 3=PC3; 4=PC2. The likely reasons for such a behaviour are the oxidation of PC and the fact that the complex formed with the oxidized form, if any, is much weaker than that with the reduced form.The formation of a diVerent complex is evident judging from the shift of the elution volume of PC2. There is also evidence for the formation of an aggregate with an apparent molecular mass of 6800 Da as seen in Fig. 2(c). This suggests that PCs may aggregate under some conditions and there is a risk of misidentification of chromatographic signals if judged from the elution volume only.Optimization of the separation conditions Because of their strongly hydrophilic anionic character, PC–metal complexes cannot be retained on a reversed-phase column even in organic media (methanol, acetonitrile). The use of strong anion exchangers leads to the need for concentrated buVers which would adversely aVect ICP-MS or ESIMS/ MS detection. The size-exclusion mode was therefore selected as the chromatographic separation mechanism.An earlier study showed the possibility of eluting Cd and Cu species in plant cytosol extracts from a size-exclusion column (Eurogel GFC, 300×7.5 mm id) as a fairly broad single peak corresponding to a compound with an apparent molecular mass of ca. 13 kDa,16 but no optimization of the separation conditions was carried out because of the non-availability of standards.16 In this work, the synthesis and purification of individual Cd–PC species allowed the systematic optimization of the SEC-ICP-MS conditions and investigation of the potential of this technique for the determination of Cd complexes with oligopeptides in plant cell extracts.Fig. 2 SEC-ICP-MS chromatograms obtained for the PC–Cd stan- Choice of the column. The SEC columns examined in this dards synthesized from individual purified PC preparations. work contained the same type (from the chemical point of (a) (GluCys)2Gly (PC2); (b) (GluCys)3Gly (PC3); (c) (GluCys)4Gly view) of stationary phase (cross-linked agarose–dextran) but (PC4); (d) mixture of PC2, PC3 and PC4 prior to separation of the diVered in terms of the exclusion limit and the optimum individual PCs equilibrated with Cd.The dashed lines correspond to the molecular masses found by the calibration of the columns as separation range. Superdex-75 oVers an eVective separation described under Analytical procedure. Peaks: 1=unidentified; 2= range (for globular proteins) of 0.5–80 kDa which has been PC4; 3=PC3; 4=PC2.judged the most suitable for the separation of metal complexes with metallothioneins so far.24 Since PC peptides are much smaller than metallothioneins, another column (Superdex prepared from a mixture of apo-PCs and one stored for 12 d at 4 °C. It is evident that solutions of standards and sample Peptide HR), designed for the separation of amino acids and oligopeptides, was investigated in this work, despite the lack extracts should be prepared fresh since storage adversely aVects the intensity of Cd–PC signals, especially for PC2 and PC3.of reports of its application to the separation of metal com- 1560 J. Anal. At. Spectrom., 1999, 14, 1557–1566plexes. A guard column filled with a packing with an exclusion limit of 80 kDa was used. The separation eYciency was investigated for a mixture of the PC2–4–Cd complexes prepared as described in the under Analytical procedures. No diVerence in terms of the separation eYciency between the Superdex 75 and Superdex Peptide columns was observed. The duration of the run was slightly shorter (by ca. 2 min) using a Peptide HR column, so this was chosen for further work. EVect of pH. Fig. 4 shows that the pH of the mobile phase aVects not only the separation eYciency but also the morphology of the chromatogram. The eVect was tested in the pH range 6.0–10.0 adjusted with 30 mM TRIS–HCl or TRIS– ammonia buVer. Lower pH values were not investigated because the PC–Cd complexes are known to be unstable in acidic media.4 At pH 6.0 the signal from PC2 is already suppressed, probably because of the dissociation of the complex and sorption of the Cd2+ on the column stationary phase.Chromatograms at pH 7.5 and 8.5 are almost identical. The separation between PC2 and PC3 is slightly better at pH 7.5, but on the other hand the run at pH 8.5 is faster by 2 min. Both pH values were examined further for the chromatography of plant cell extracts. At pH 10.0 all the cadmium elutes as a single compound in the void volume of the column.The total cadmium eluted (calculated from the area under the peak) is similar to that at pH 7.5 and 8.5. It is therefore concluded that another compound is formed with PC2, PC3 and PC4 as ligands. This corroborates the observation of Grill et al.26 who predicted, under some conditions, the formation of Cd–thiol complexes consisting of a mixture of the PC peptides of various lengths with small amounts of cysteine and glutathione.EVect of buVer concentration. Fig. 5 shows that the concentration of the buVer in the mobile phase does not play an important role provided that it is not lower than 10 mM. In the absence of salt the elution eYciency is fairly low, and all the cadmium elutes as a single peak in the void volume of the column. This is not the eVect of the pH since at the same pH in the presence of buVer a ‘normal’ elution pattern was observed [cf., Fig. 4(a)].The chromatogram in Fig. 5(a) suggests that in the absence of salt aggregated species are formed. They are apparently less stable than the complexes and the exchange of the cadmium with the column stationary phase occurs more readily. At elevated buVer concentrations (70 mM) the baseline is higher, probably because of the column bleeding with Cd2+. Figures of merit The precision of the method was evaluated by fivefold injection of a mixture of Cd–PC2, –PC3 and –PC4 complexes at the 1 mg ml-1 level (as PC) and the determination of the peak area. The relative standard deviations were 4.7, 4.6 and 6.9% for Cd–PC2, –PC3 and –PC4, respectively.Fig. 4 EVect of pH on the separation of PC–Cd complexes by SEC. The linearity of the PC peak area as a function of concen- ICP-MS detection of cadmium. Amount injected, 1 mg ml-1 (as PC). tration was investigated in the range 0.01–15 mg ml-1 of PC pH: (a) 6.0; (b) 7.5; (c) 8.5; (d) 10. Peaks: 1=unidentified (void volume of the column); 2=PC4; 3=PC3; 4=PC2.in the chromatographed solution. Calibration curves are shown in Fig. 6. Two linearity ranges can be distinguished; a well marked break point is observed around concentrations of 2–3 mg ml-1 for each of the analysed PCs. The detection limits cadmium from the injected PC–Cd complex by other metals present on the column cation-exchange sites was investigated. (calculated as three times the baseline noise) are at the 10 ng ml-1 level for each of the phytochelatins.The approach undertaken consisted of injecting a metal-free PC solution and monitoring the chromatographic eluate for The reason for such an anomalous behaviour of the calibration curve seems to be due either to the formation of the presence of metal complexes by ICP-MS. It was found that Cd and Cu were present in the eluate, diVerent Cd complexes as a function of the PC concentration, or to a reaction taking place on the column. Since the giving rise to signals matching the elution volumes corresponding to those of the PC–Cd complexes.The intensities of these morphology of the chromatogram was found to be independent of the PC concentration injected, the second reason seems signals were random and depended on the history of the column. When a PC–Cd standard was injected, intense signals to be more likely. The possibility of the displacement of J. Anal. At. Spectrom., 1999, 14, 1557–1566 1561Fig. 6 Calibration curves for the PC–Cd complexes.Calibration curves for the lower concentration range are shown in the inset. (a) (GluCys)2Gly (PC2); (b) (GluCys)3Gly (PC3); (c) (GluCys)4Gly Fig. 5 EVect of the TRIS–HCl buVer concentration on SEC of PC–Cd (PC4). complexes. ICP-MS detection. Amount injected, 0.1 mg ml-1. BuVer concentration: (a) none (water at pH 6.0); (b) 10 mM; (c) 30 mM; (d) 70 mM. Peaks: 1=unidentified (void volume of the column); 2= intensity) and (iii) Cu2+ adsorbed on the column exchanges PC4; 3=PC3; 4=PC2.with Cd from a PC–Cd complex leading to the destruction of the latter. It was therefore decided to develop a protocol for column conditioning in order to be able to obtain reproducible were observed on the Cu channel, sometimes in addition to chromatograms. the Cd signals but often instead of Cd signals. When a Cu2+ solution was injected on to the column the following chromato- Column conditioning gram of a PC–Cd standard showed a distorted morphology.It was concluded that (i) metal ions (Cd, Cu and to some The most straightforward solution to the problem of artefact formation consists of preventing the free Cu2+ and Cd2+ ions extent Zn) accumulate on the column until an equilibrium is reached, (ii) these metals may be complexed by free ligands from accumulating at the head of the column. A similar problem was solved in the case of metal complexes with present in the injected solution giving rise to ghost signals (or to signals from analyte compounds but of uncontrollable metallothioneins by adding b-mercaptoethanol to the injected 1562 J. Anal.At. Spectrom., 1999, 14, 1557–1566solution.30 This chelating compound complexed Cu2+ and TRIS–HCl buVer (pH 7.5) and 30 mMacetate buVer (pH 7.0). No diVerence in terms of the morphology of the SEC-ICP-MS Cd2+ ions without aVecting the complexation equilibrium of the analyte species (MT–Cd), which allowed the elution of chromatograms of these extracts was observed.The buVer was used for extraction of metal–peptide complexes in order to the free metal ions as a peak in the total volume of the column, after the peak of the analyte compound. In the case of PC ensure reproducible extraction conditions in terms of pH. Chromatograms obtained under the optimized analytical complexes it turned out, however, despite some reports claiming the stability of PC–metal complexes in the presence of conditions (Peptide HR 10/30 column) for the above samples are shown in Fig. 8. The intensity of the signals was found to b-mercaptoethanol,16 that b-mercaptoethanol competes with the PC ligands, leading to the destruction of the PC–Cd decrease even after 1 d of storage of the extract but no changes in the morphology of the chromatogram were observed. complexes. An alternative is the cleaning of the column by repetitive Chromatography at pH 7.5 was verified and was found to give very similar chromatograms (not shown). injections of b-mercaptoethanol to remove the metals accumulated in the previous run.The advantages are the ease of The extract of Silene cucubalus contains Cd complexes of PC2, PC3 and PC4 as confirmed by the matching elution control of the eluted metal (peak intensity monitored in real time by ICP-MS) and the lack of need to change the mobile volumes of the appropriate standards. PC2, PC3 and a trace of PC4 were found in Rauwolfia serpentina extract. PC2 and phase between sample injections.However, whereas two injections of the reagent solution are suYcient to remove Cd from PC3 signals were also identified in Agrostis tenuis extract. The intensity of Cd complexes in the maize samples is much lower. the column, a large amount of Cu2+ stays on the column even after the fourth injection of b-mercaptoethanol (Fig. 7). The Nevertheless, a distinct PC2 signal can be identified. Note that Silene and Agrostis extracts were diluted for the analysis.only possibility therefore seems to be the introduction of a regular washing step between the injections. The most promising results in terms of reproducibility (ca. 5% as indicated above) were obtained by conditioning the column between the injections with a 2 mMb-mercaptoethanol solution in 30 mM TRIS buVer (pH 7.5) for 30 min. The removal of the chelating agent prior to next injection was accomplished by conditioning the column with the mobile phase for another 30 min.Care should be taken to avoid traces of metals in the conditioning solutions, otherwise trace metals will accumulate at the head of the SEC column during the conditioning phase. A sorption column removing trace metals (especially Cu) at the exit from the pump is recommended. 31 The state of the column should be controlled prior to introduction of a sample by injection of a b-mercaptoethanol solution. Typical peak height values were lower that 200 counts s-1 for Cd and 500 counts s-1 for Cu.The cleanliness of the column aVects the slope of the calibration curve in the lower concentration range and the position of the break point on the latter. Analysis of plant extracts Detection of metal–peptide complexes in plant extracts. The method developed was applied to speciation of cadmium in extracts of a plant, maize (Zea mays L.), and of three diVerent plant cell cultures of Silene cucubalus, Agrostis tenuis and Rauwolfia serpentina, known to biosynthesize PCs when exposed to Cd2+.The procedures investigated to extract the Cd complexes included extraction with water, 30 mM Fig. 8 SEC-ICP-MS chromatograms of plant extracts prepared according to the Analytical procedure. (a) Silene cucubalus; (b) Agrostis tenuis; (c) Rauwolfia serpentina; (d) maize (Zea mays L.). Fig. 7 EYciency of the post-run removal of Cd (&) and Cu (#) from Peaks: 1=unidentified (void volume of the column); 2=unidentified (Mr 6800); 3=PC4; 3=PC3; 4=PC2. The dashed vertical lines corre- the column by repetitive injections of b-mercaptoethanol. Mobile phase, water (open symbols) and 30 mM TRIS buVer (pH 8.5) spond to the molecular masses found by the calibration of the columns as described under Analytical procedure.(closed symbols). J. Anal. At. Spectrom., 1999, 14, 1557–1566 1563In addition to the above discussed signals for which identifi- the careful column conditioning but the regular washing of the column makes it occur in a reproducible way.It should cation was possible on the basis of their elution volumes matching those of the previously synthesized and characterized also be noted that the amounts of Cd2+ and that of free PC ligands in the injected solutions are actually unknown. It is PC2–4–Cd standards, at least one (sometimes two) intense Cd signals are observed in the SEC-ICP-MS chromatograms of therefore impossible to evaluate whether this apparently low recovery is due to the decomposition of the complex on the the extracts investigated. All the extracts contain a compound with an apparent molecular mass of 6800 u similar to that column or simply because only 50% of Cd in the injected solution is actually complexed.The second possibility is cor- already observed in the isolated PC4 standard [Fig. 2(c)]. Moreover, the extracts show the presence of a Cd species roborated by the fact that chromatography is reproducible, the baseline is low and stable and the chromatographic peaks excluded from the column.The chromatograms in Fig. 8 exhibit a diVerent morphology do not tail. in comparison with the only chromatogram reported so far, to our knowledge, in the literature and obtained by a similar Quantification of PC–Cd complexes in plant extracts. The technique. Leopold et al.,3,16 analysing an extract from Silene above data indicate that Cd occurs in the extracts of plants in vulgaris by SEC-ICP-MS, showed that cadmium eluted as a a number of fairly labile complexes.This makes the approfairly broad single peak at an elution volume corresponding priate calibration a critical issue. Attempts to use the method to an apparent molecular mass of ca. 13 kDa. Since no of standard additions instead of the calibration curve failed calibration standards were available, this compound was ident- since the addition of a standard modified the equilibrium in ified as a Cd, Cu–PC2 complex on the basis of the detection the system, leading to changes in the mutual ratio of the of PC2 and desglycyl-PC by ESI-MS.3 In order to obtain a diVerent Cd–PC complexes present.External calibration using deeper insight into the identity of the unknown signals in the a calibration graph established as in Fig. 6 was therefore used. chromatograms in Fig. 8, two approaches were used in this The results obtained for the diVerent samples analysed are work. The extracts were chromatographed on size-exclusion summarized in Table 2.Note that, in contrast to the injection columns with a higher exclusion limit to determine more of the standard solution, the average precision of the determiprecisely the apparent molecular mass of the early eluted nation of PC–Cd complexes in real samples is ca. 20%, which compounds. Further, the fractions containing the unknown is probably a consequence of a number of dynamic equilibria peaks were collected and characterized by ESI-MS-MS. occurring in the system.Signal identification in SEC-ICP-MS chromatograms of plant Validation of the method developed. Since reference materials extracts. Chromatograms (not shown) obtained using the with known PC concentrations in plant samples are not Superdex 75 column (exclusion limit 100 kDa) and Superdex available, the only way to validate the method developed was 200 (exclusion limit 1300 kDa) for the plant extracts analysed to compare the results obtained with those obtained by an in Fig. 8 always show a signal excluded from the column that independent analytical method. No other method is available, corresponds, judging from the morphology of the chromatog- however, for the determination of Cd–PC species. Therefore, rams, to the first eluting peak in Fig. 8. The fraction containing the only possibility to validate the method developed was to this peak collected and analysed by ESI-MS did not show any compare the concentrations of the individual PC ligands found trace of PCs.A possible hypothesis of the identity of this peak by SEC-ICP-MS with those obtained by reversed-phase HPLC is that it contains a complex of cadmium with polymers of with post-column derivatization with 5,5¾-dithiobiscell membranes which were not completely removed by ultra- (2-nitrobenzoic acid) and spectrophotometric detection. The centrifugation. latter measurements were carried out in a diVerent laboratory The second of the unknown signals in Fig. 8 behaves on the by a diVerent operator. The agreement is satisfactory but, in Superdex 75 and Superdex 200 columns like a compound with general, the results obtained by SEC-ICP-MS are 20–30% an apparent molecular mass of ca. 10 kDa. The fraction lower. This may be due to the fact that the PCs present in the containing this peak collected, desalted on a reversed-phase peak 2 in Fig. 8 are not taken into account in the SECcolumn as described in the under Analytical procedure and ICP-MS procedure.Note that the ratio of Cd to PC for peak analysed by ESI-MS produced the spectrum shown in Fig. 9. 2 seems to be much higher than that for the PC2, PC3 and It shows at least three peaks, at m/z 540, 772 and 1004, which PC4 complexes. may correspond to the protonated molecular ions of PC ligands. Indeed, the product ion spectra of the three peaks (not shown) confirmed the presence of amino acid sequences Conclusions of PC2, PC3 and PC4. The data shown in Fig. 9 were obtained SEC with ICP-MS detection is a valuable tool for screening with Silene cucubalus. Similar spectra were obtained for the for metal complexes in biological tissue cytosols, but its proper other samples investigated, except maize, for which the ESI use requires a systematic approach to each problem to be mass spectrum was hardly diVerent from that of the blank, solved in terms of standardization and verification of the probably because of the 100-times lower concentration of the stability of the species studied.In contrast to organometallic species of interest. compounds, metal coordination complexes are likely to undergo metal (ligand) exchange either with the metal or Recovery. Recovery of Cd from the column was evaluated ligand contaminants retained at the head of the column or by comparing the amount of the metal injected with that with the column active sites themselves. Nevertheless, when a eluted. The results are given in Table 1.The average recovery number of precautions are taken, the SEC-ICP-MS method of Cd oscillates around 50% with a trend of being lower for allows not only the qualitative analysis of heavy metal species more dilute solutions. This corresponds well with the lower in plants but also their quantitative determination. slope of the calibration curve in the low concentration range (cf., Fig. 6). One of the reasons for such a low recovery may be the exchange of the Cd from the PC–Cd complex with Acknowledgements Cu2+ present on the column.Indeed, the results shown in Table 1 demonstrate that a significant amount of copper is The authors thank Professor Dr. M. Zenk and Matjaz Oven (University of Munich) for valuable discussions. Dr. released from the column which can only occur by complexation with a PC ligand. This phenomenon occurs despite R. Lobinski is acknowledged for critical reading of the manu- 1564 J. Anal. At. Spectrom., 1999, 14, 1557–1566Fig. 9 ESI tandem mass spectra of peak 2 in Fig. 8. Data are shown for Silene cucubalis. Peaks: 1=PC2; 2=PC3; 3=PC4. Table 1 Recovery of cadmium and copper in SEC for standards and sample extracts Metal injecteda/ Metal recovered/ Recovery (%) ng ng Sample analysed Cd Cu Cd Cu Cd Cu Apophytochelatin (a-PC), 1 mg ml-1 <0.1 <0.1 <0.1 0.5 — — a-PC+0.1 mg Cd 3 0.1 0.6 0.5 20 500 a-PC+0.25 mg Cd 7 0.2 2 0.9 29 450 a-PC+0.5 mg Cd 14 0.2 5 0.2 36 100 a-PC+1 mg Cd 25 0.1 10 0.4 40 400 Silene vulgaris 11 0.1 5.5 1.1 51 1100 Silene cucubalis 26 0.1 10 1.1 39 1100 Agrostis tenuis 30 0.1 18 1.0 60 1000 Rauwolfia serpentina 71 0.4 30 1.6 43 400 Maize 8.5 0.2 2.7 1.7 32 850 aDetermined by ICP-MS in the injected solution.script. The work was financed by grant No. 130201 Z0025 (Region Aquitaine–FEDER). 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