This focal point review provides an overview of recent developments and capabilities of inductively coupled plasma mass spectrometry (ICP-MS) coupled with different separation techniques for applications in the fields of quantitative environmental and bio-analysis. Over the past years numerous technical improvements, which are highlighted in this review, have helped to promote the evolution of ICP-MS to one of the most versatile tools for elemental quantification. In particular, the benefits and possibilities of using state-of-the-art hyphenated ICP-MS approaches for quantitative analysis are demonstrated with a focus on environmental and bio-analytical applications.

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A comparison of different nebulisers for direct hyphenation of capillary and nano liquid chromatography (Cap-LC, Nano-LC) and quadrupole-based collision cell inductively coupled plasma mass spectrometry (CC-ICP-MS) for phosphorylation profiling of tryptic protein digests is described. Helium was used as cell gas and specially tuned instrumental conditions were used to achieve background minimisation at the mass of phosphorus, because of kinetic energy discrimination of the interfering polyatomic ions. The proposed set-up is based on a modified capillary electrophoresis interface and a home-made 4 mL spray chamber. It enables the use of gradient conditions with a highly concentrated organic mobile phase as often used in protein phosphorylation analysis, without the need to apply membrane desolvation for removal of the organic phase or further background minimisation. No significant signal suppression or other negative effects caused by the organic mobile phase occur, because of the low flow rates used in Cap-LC and the robust plasma conditions of the CC-ICP-MS instrument. A tryptic digest of beta-casein was investigated as model compound to demonstrate the applicability of the proposed set-up for phosphorylation profiling in protein analysis using quadrupole based collision-cell ICP-MS as phosphorus-specific detector. Detection limits for phosphorylated peptides down to the sub picomole level were obtained. As a complementary technique, electrospray ionisation tandem mass spectrometry (ESI-MS-MS) with data base searching was used for further characterisation of the phosphorylated peptides detected.

A new approach for the speciation of metallothioneins (MT) in human brain cytosols is described. The analysis is performed by application of a newly developed coupling of capillary electrophoresis (CE) with inductively coupled plasma-sector field mass spectrometry (ICP-SFMS). Isoforms of metallothioneins are separated from 30-100 microliter sample volumes by CE and the elements Cu, Zn, Cd, and S are detected by use of ICP-SFMS. The extraction of cytosols is the first step in the analytical procedure. Tissue samples from human brain are homogenized in a buffer solution and submitted to ultra-centrifugation. The supernatant is defatted and the cytosol pre-treatment is optimized for CE separation by matrix reduction. The buffer concentration and pH used for capillary electrophoretic separation of metallothionein from rabbit liver were optimized. CE with ICP-MS detection is compared to UV detection. In the electropherograms obtained from the cytosols three peaks can be assigned to MT-1, MT-2, and MT-3. As an additional method, size-exclusion chromatography (SEC) is applied. Fractions from an SEC separation of the cytosol are collected, concentrated, and then injected into the CE. The detection of sulfur by ICP-SFMS (medium resolution mode) and quantification by isotope dilution have also been investigated as a new method for the quantification of MT isoforms. The analytical procedure developed has been used for the first time in comparative studies of the distributions of MT-1, MT-2, and MT-3 in brain samples taken from patients with Alzheimer's disease and from a control group.

In a new approach to the characterization and quantification of metallothionein isoforms an on-line isotope-dilution method in combination with the coupling of capillary electrophoresis (CE) to an inductively coupled plasma-sector field mass spectrometer (ICP-SFMS) is reported. Metallothionein (MT) isoforms are separated by CE and the elements Cu, Zn, Cd, and S are detected simultaneously by use of ICP-SFMS in the medium resolution mode. On-line isotope dilution is performed by continuous introduction of an isotopically enriched, species-unspecific spike solution after the separation step. MT from rabbit liver and a further purified MT-1 isoform were quantified by determination of sulfur, and the stoichiometric compositions of the metalloprotein complexes are characterized by determination of their sulfur-to-metal ratios.

Recent developments in the coupling of highly selective separation techniques such as capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) to element-specific and molecule-specific detectors, such as inductively-coupled plasma mass spectrometry (ICP-MS) and electrospray ionization-tandem mass spectrometry (ESI-MS/MS) for the characterization and quantification of metallothioneins (MTs) are critically reviewed and discussed. This review gives an update based on the literature over the last five years. The coupling of CE to ICP-MS is especially highlighted. As a result of progress in new interface technologies for CE-ICP-MS, research topics presented in the literature are changing from "the characterization of interfaces by metallothioneins" to the "characterization of metallothioneins by CE-ICP-MS". New applications of CE-ICP-MS to the analysis of MTs in real samples are summarized. The potential of the on-line isotope dilution technique for the quantification of MTs and for the determination of the stoichiometric composition of metalloprotein complexes is discussed. Furthermore, a selection of relevant papers dealing with HPLC-ICP-MS for MT analysis are summarized and compared to those dealing with CE-ICP-MS. In particular, the use of size-exclusion (SE)-HPLC as a preliminary separation step for metallothioneins in real samples prior to further chromatographic or electrophoretic separations is considered. Additionally, the application of electrospray ionisation-tandem mass spectrometry (ESI-MS/MS) for the identification of metallothionein isoforms following electrophoretic or chromatographic separation is discussed.