Simultaneous Speciation of Selenoproteins and Selenometabolites in Plasma and Serum.

Selenium is an essential element incorporated to different proteins with important biological functions in connection to antioxidant activity, cancer-protective properties, neurodegenerative pathologies, and prevention of effects of diabetes, among others. In addition, selenoamino acids play a basic role in the global equilibrium of key selenium-biomolecules synthesis, including selenoprotein P, selenoalbumin, and glutathione peroxidase. Homeostasis of these selenium-containing biomolecules involves different organs in living organisms including human, and bloodstream is the connection fluid in this process. Therefore, it is very important to have an analytical methodology suitable for selenium proteins and metabolites speciation in serum and plasma samples. For this purpose, a simultaneous speciation method for Se-containing biomolecules in serum/plasma is described on the basis of in series three-dimensional chromatography: size exclusion, affinity, and anion exchange high performance liquid chromatography (3D/SE-AF-AEC-HPLC), using different columns of each type and hyphenation to inductively coupled plasma-(quadrupole) mass spectrometry (ICP-MS). The method allows the quantitative simultaneous analysis of selenoprotein P (SeP), extracellular glutathione peroxidase (eGPx), selenoalbumin (SeAlb), selenite, and selenate in serum (from human and mouse) using species-unspecific isotope dilution (SUID). In addition, a simplified two-dimensional approach (2D/SE-AF-HPLC-SUID-ICP-MS) is described when selenium metabolites are globally analyzed. The method provides detection limits in the range 0.2-1.3 ng of Se g and avoids typical interferences in this matrix from chloride and bromide with a chromatographic runtime less than 35 min.