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International audienceAn analytical strategy has been developed for the characterization of Cd complexes in rat liver tissue. The sample preparation included the isolation of the Cd-macromolecular fraction by semi-preparative size-exclusion chromatography, followed by a preconcentration step by lyophilization and desalting. The analysis was carried out by microbore reversed-phase HPLC with parallel ICP-MS and ESMS detection. The HPLC-ICP-MS interface was based on a Micromist nebulizer and a low dead volume cyclonic spray chamber. The HPLC-ESMS interface allowed the measurement of the molecular mass of the ligand protein owing to the dissociation of the complex by the on-line post-column acidification of the eluate. The approach allowed the detection of two major metallothionein (MT) isoforms and their identification on the basis of the molecular mass as rat liver MT-1 and MT-2. The actual number of peaks in the chromatograms was larger because of the formation of mixed Cd-Cu complexes of the same MT isoform that showed different hydrophobicities

An interface between capillary zone electrophoresis and inductively coupled plasma mass spectrometry (CZE-ICP-MS) based on a self-aspirating total consumption micronebulizer was evaluated for a study of metal complexation by metallothionein. The elimination of the nebulizer suction (and thus of the laminar flow) allowed a separation efficiency comparable with that reachable with on-capillary UV detection whereas the low (6 ml min 21 ) nebulizer aspiration rate allowed the minimization of the post-capillary dilution effect, and thus maximization of the CZE-ICP-MS sensitivity. Operating conditions were optimized with the natural rabbit liver metallothionein preparations allowing detection limits of ca. 10 ng mL 21 (as MT-bound Cd) to be obtained within 10 min at quasi-baseline resolution. Recombinant (mouse liver MT-1-Zn 7 ) and natural (rabbit liver MT-1-Cd 7 and MT-2-Cd 7 ) proteins were then titrated with Cd(II) and Cu(I), and Cu(I), respectively. A number of mixed metal complexes with different migration times were observed as a function of the Cu : Cd ratio in the system.