Signal identification in size-exclusion HPLC-ICP-MS chromatograms of plant extracts 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 heartcut, purified by reversed-phase chromatography, and analysed by ES-MS/MS. The purity and identity of signals in ES-MS/MS spectra were further confirmed by collision induced dissociation (CID)-MS. The different peaks in SEHPLC-ICP-MS turned out to contain identical ligands at different ratios. On the other hand, a single SEHPLC-ICP-MS chromatographic signal was demonstrated to contain a number of complexes with ligands varying in terms of amino-acid chain length, amino-acid sequence and stoichiometry. Whereas the particular ligands (phytochelatins modified on the C-terminal amino-acid) could 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 of peaks is demonstrated.