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 µg 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 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.

Speciation of cadmium in plant tissues by size-exclusion