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A capillary zone electrophoresis (CZE) method with preceding cationic transient capillary isotachophoresis (tCITP-CZE) was developed for uncoated fused-silica capillaries to analyze metal-binding proteins (MBPs) of clinical relevance. UV detection was followed by mass spectrometry (MS). Optimization was done with model proteins of properties similar to relevant human MBPs. Using 1.0 mol x L(-1) formic acid (pH 1.78) as electrolyte resulted in up to 165000 plates m(-1) in CZE and 230000 plates m(-1) in combination with tCITP and analysis time was less than 5 min in uncoupled mode. Cationic tCITP with 125 mmol x L(-1) ammonium formate, buffered to pH 4.00, as leading electrolyte improved sample loadability considerably in comparison with sample stacking without impairing resolution. Following systematic optimization of the electrospray ionization process (ESI) the coupled system ((tCITP)-CZE-UV-ESI-MS) was tested with protein model mixtures and human MBPs. Repeatability of migration times was < 0.64% in pure CZE mode and in tCITP-CZE mode and < 0.83% in CZE-ESI-MS coupled mode. Mass accuracy was < 0.015%. Limits of detection were found to be in the range 50-160 fmol.

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A robust, user-friendly, sensitive and affordable speciation method based on microbore anion-exchange chromatography (IC) for the separation of selenium species and Zeeman-effect ETAAS for element specific detection is described. By exploiting the very low flow rates normally employed in microbore chromatography, the analytical usefulness of chromatography coupled to the sensitive but discontinuous ETAAS detector has been increased in comparison with couplings incorporating normal bore liquid chromatography. The flow rate of the mobile phase in this particular IC system was 80 ml min 21 . A highly reproducible and automated collection of 20 ml fractions in sampler cups was used for interfacing the chromatographic separation with ETAAS. The IC-ETAAS results were also directly compared with those obtained by IC-direct injection nebulizer ICP-AES for assessing the chromatographic resolution of the proposed method. It is possible to separate selenomethionine, selenite, selenate and selenocystine in 6 min. The trade-off in chromatographic resolution and time consumption in the detection step using IC-ETAAS is compensated for by a high degree of simplicity and the high specificity and sensitivity of the Zeeman-effect ETAAS resulting in relative detection limits of 2.8-4.1 ng ml 21 (42-61 pg absolute). These detection limits are comparable to those for published HPLC-ICP-MS methods. The method was evaluated by injecting aqueous standards, unspiked and spiked sample extracts from the biological certified reference material CRM 402. The eluent was injected along with a palladium-magnesium nitrate modifier since the pyrolysis curves for trimethylselenonium, selenomethionine, selenite, selenate and selenocystine revealed a similar response for all species by using this modifier, i.e, all species are thermally stabilized up to 1000 °C. Without adding the modifier, the species showed very different volatilities during the thermal pre-treatment step.