Signal identification in size-exclusion HPLC-ICP-MS chromatograms of plantextracts by electrospray tandem mass spectrometry (ES MS/MS)

Pneumatically-assisted electrospray (ion-spray(R)) tandem mass spectrometry (ES-MS/MS) was evaluated for the identification of cadmium binding ligands in complexes detected in extracts of plant cell cultures by size-exclusion HPLC (SEHPLC) with ICP-MS detection. The metal-containing fractions were h eartcut, purified by reversed-phase chromatography, and analysed by ES-MS/M S. The purity and identity of signals in ES-MS/MS spectra were further conf irmed by collision induced dissociation (CID)-MS. The different peaks in SE HPLC-ICP-MS turned out to contain identical ligands at different ratios. On the other hand, a single SEHPLC-ICP-MS chromatographic signal was demonstr ated to contain a number of complexes with ligands varying in terms of amin o-acid chain length, amino-acid sequence and stoichiometry. Whereas the par ticular ligands (phytochelatins modified on the C-terminal amino-acid) coul d be identified, the complexity of equilibria within the isolated fraction prevented the elucidation of the stoichiometry of the complexes formed. The results question the validity of signal identification in SEHPLC-ICP-MS by elution volume matching with standards unless the chromatographic purity o f peaks is demonstrated.