IntroductionMetallospecies play a unique and important role in many essential life processes of which the mechanisms are still poorly understood at the molecular level. Therefore, the detection, identification and determination of trace element compounds in biological systems—bioinorganic speciation analysis—has been enjoying a lot of interest in recent years, becoming an emerging field of analytical chemistry.1–4In this context, particular attention has been given to hyphenated techniques that combine the high separation efficiency of high-performance liquid chromatography (HPLC) or capillary zone electrophoresis (CZE) with the elemental or molecular specificity of inductively coupled plasma (ICP) or electrospray (ES) mass spectrometry (MS), respectively.5The characterization of metal complexes with metallothionein, a low molecular mass (6–7 kDa) metal-binding protein, synthesized by mammals in response to heavy metal stress,6,7has been one of the most popular topics investigated by hyphenated techniques. Liquid chromatography followed by metal-specific detection by ICP-MS was widely applied to the detection and determination of MT and its isoforms in animal tissues.8–16The high separation efficiency of CZE for metal-binding peptides17–19has made many researchers choose rabbit liver metallothionein (MT), preparation available from Sigma–Aldrich, as the favourite test sample to demonstrate the performance of newly developed CZE-ICP-MS interface designs.20–23The recombinant mouseliver MT-1 was recently used for a study of metal complexation equilibria by CZE-ICP-MS.24However, no applications of CZE-ICP-MS to the characterization of metal complexes in animal tissues cytosols have been developed so far.The to-date studies of bioinduction of MTs in rat by hyphenated techniques were limited to Zn-induced proteins, which allowed an easy demetallation of MTs prior to their characterization as apo-forms by CZE-MS.25,26The acidic buffer conditions employed for the separation did not allow the investigation of the complexes present since the link between the metal and the ligand in the complex had been destroyed.25–27Our attempts to use this approach for the characterization of Cd-induced MTs turned out to be unsuccessful because of the impossibility of efficient demetallation of Cd–MT with CZE acidic buffers.28In another approach, signals of major MT-1 and MT-2 isoforms could be obtained for rat tissues but the signal to noise ratio was poor.29Most of information acquired on MT by hyphenated techniques so far has been qualitative. It was usually limited to the number of peaks present in the chromatogram or electropherogram and sometimes included identification data from mass spectrometry. The quantitative aspect was usually neglected. In the best case, the quantification was carried out on the basis of a tentatively assigned complex stoichiometry. Recently, Schaumlöffelet al.proposed determination of MT based on the on-line determination of sulfur in CZE peaks by ICP-SF-DF-isotope dilution-MS.30The objective of this study was the evaluation of ICP-sector field-MS and isotope dilution quantification for the characterization of metal complexes with MT induced in the liver of a rat exposed to Cd stress. Our particular attention was focused on the MT quantification, the determination of the stoichiometry of the complex, and on the complementarity of electrospray MS for the identification of the MT isoforms present.