Speciation of cadmium in plant tissues by size-exclusion chromatography with ICP-MS detection

Abstract

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 (PC 2 ), (GluCys) 3 Gly (PC 3 ) and (GluCys) 4 Gly (PC 4 ) 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 PC 2 , PC 3 and PC 4 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 different 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.

An HPLC-ICP-MS technique for determination of cadmium–phytochelatins in genetically modified Arabidopsis thaliana

“…Cadmium-phytochelatin (Cd-PC) complexes are strongly hydrophilic and anionic in nature and thus will not be retained in a reversed-phase column as such, even in the presence of organic modifiers [26]. Moreover, trifluoroacetic acid, acetic acid or formic acid (at concentrations ≤0.1%) used in the mobile phase in a typical reversed-phase separation can dissociate the Cd-PC complexes as they are known to be unstable at lower pH values.…”

“…The oxygen flow was reduced until a buildup of carbon on the sampling cone was observed. The oxygen flow was then increased until the carbon deposits were decomposed and the green C2 emission "tongue" visible in the central channel of the plasma was seen to stop well before the sample cone orifice [26].…”

“…After extraction the solution was ultra-centrifuged (Beckman Coulter, Fullerton, CA, USA) for 30 min at 10 000 × g at 4 °C and filtered through a 0.45 μm syringe filter. The filtrates were injected on the HPLC column with 10 times dilution using mobile phase solvent A. Cadmium-phytochelatin complexes (Cd-PC2, Cd-PC3, and Cd-PC 4 ) were used as standards for HPLC-ICP-MS method development and were prepared following the method of Vacchina et al [26].…”

“…An SEC-ICP-MS technique developed by Vacchina et al showed, for the first time, the separation of individual cadmium-phytochelatin standards with satisfactory figures of merit and was applied to cadmium-phytochelatin speciation in plant tissue [26]. However, the residual anionic charges on the stationary phase of the size-exclusion column compete for the metal ions from the cadmium-phytochelatin complexes and they are irreversibly adsorbed onto the stationary phase.…”

Metal and metalloid speciation in plants: Overview, instrumentation, approaches and commonly assessed elements

“…Such analyses can be described as the identification and measurement of specific forms of an element according to its molecular or complex structure, electronic or oxidation state or its isotopic composition [8]. The interest in metal and metalloid species in plants (which are generally collectively referred to as "metals" in this paper) is well established, as plants are natural, dietary sources for major, minor and trace essential minerals [9]. Since metals are nonbiodegradable [10; 11], metal loading in plants is therefore an important consideration when assessing potential health impacts [12; 13], where metals can be assimilated either as elements or as metabolites [14].…”