speciation of selenium in human biological samples using online hyphenation of HPLC and ICP-MS

Selenium is an important trace element that plays specific biological role in organism. Since bioactivity of selenium is a consequence of individual chemical species, there is considerable interest in the speciation of selenium in the mammalian organism. Inasmuch as selenium is primarily present in the form of selenoproteins in mammalian tissues, speciation studies of Se-containing proteins are focused on the various organs and tissues. Serum and urine samples can be used for study of Se metabolites in vivo. Hyphenation of chromatographic separation with element-specific detection has been proved as a powerful technique for Se speciation analysis recently. In present study, using an ion-pair reversed-phase liquid chromatography, Se species such as selenite (Se(IV>), selenate (Se(VI>), trimethyl selenonium (TMSe+), selenocystine (SeCys), selenourea (SeUr), selenomethionine (SeMet), and selenocystamine (SeCM) are separated and followed by the online coupling detection as introduced below. A 5-μm Symmetry Shield RP18 column was applied for the analysis of Se compounds using 0.1% HFBA in 1:99 methanol-water buffer as mobile phase. Elemental signal detected by ICP-MS geminated in case of collision cell technique (CCT) mode, since the most abundant isotope 80Se instead of 82Se or 78Se monitored in ordinary model. More sensitive technique based on ICP-MS and CCT can recognize signals of Se standard compounds, whose concentrations were less than l ppb in hyphenation experiment, and Se concentrations of quantitative analysis could reach several ppb. Human liver cytoplasm and serum samples gave legible chromatograms with a few peaks recognized easily, while urine samples of human gave much more complex chromatograms according to the intricate components. The uncertainty of measurement will be estimated too.Nevertheless, both of serum and urine samples give quantifiable elemental signals, while cytoplasm samples gave weaker signals referring to the lower concentrations of Se compounds. Hyphenated method of HPLC and ICP-MS is also a powerful technique for selenoprotein separation in biological samples. For this purpose, size exclusive chromatography (SEC) is a competent alternative, Proteins can be eluted according to their molecular weights, and selenoproteins or Se-containing proteins can be implied by the corresponding elemental signals detected by the online-coupled ICP-MS. But for an immature technique, only the evidence of Se. existence can be found. More strictly optimized conditions for analysis still remain for further study at present. The characterization of selenoprotein needs more detailed analysis, such as by 2D electrophoresis, ESI-MS and so on. More improvement is still required for this coupling technique with the progress of the analysis science.