Quantitative speciation of selenium in human serum by affinity chromatography coupled to post-column isotope dilution analysis ICP-MS
A post-column isotope dilution analysis (IDA) methodology has been applied to carry out the quantitative speciation of selenium in human serum by affinity chromatography coupled to inductively coupled plasma mass spectrometry (ICP-MS) with an octapole reaction system (ORS). The interfering argon dimers on the 78Se and 80Se isotopes were suppressed by pressurizing the octapole chamber with hydrogen. The separation of the selenium-containing proteins was evaluated both by anion exchange chromatography (Mono Q HR 5/5) and affinity chromatography (Hi-Trap Heparin- and Hi-Trap Blue-Sepharose columns). Quantification of selenium was performed by post-column isotope dilution analysis by continuous mixing of an enriched 77Se spike solution with the eluent from the column. Finally, the 78Se/77Se or 80Se/77Se isotope ratios were monitored and the amount of selenium bound to proteins was evaluated. The chromatographic separation of selenium-containing proteins in human serum by anion exchange chromatography was not satisfactory. On the other hand, the use of affinity chromatography allowed for a rapid, precise and convenient fractionation of those proteins. Three main selenium fractions could be separated and quantified: selenoprotein P, albumin and glutathione peroxidase. Mass balance performed under different experimental conditions showed quantitative selenium recovery. The proposed methodology was used to study qualitative and quantitative speciation of selenium in human serum samples from healthy volunteers and patients on haemodialysis. The distribution of selenium between plasma glutathione peroxidase (∼20%), selenoprotein P (∼55%) and albumin (∼20%) was similar in both populations.