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J Chromatogr A. 2013 Nov 29;1318:171-9. doi: 10.1016/j.chroma.2013.10.012. Epub 2013 Oct 10.

Development of a new column switching method for simultaneous speciation of selenometabolites and selenoproteins in human serum.

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Department of Chemistry and Materials Science, Faculty of Experimental Sciences, University of Huelva, Campus de El Carmen, 21007 Huelva, Spain; Research Center on Health and Environment (CYSMA), University of Huelva, Spain; International Campus of Excellence on Agrofood (ceiA3), University of Huelva, Spain.


A method for the simultaneous speciation of selenoproteins and selenometabolites in human serum has been developed 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-qMS). The method allows the quantitative simultaneous analysis of selenoprotein P (SeP), extracellular glutathione peroxidase (eGPx), selenoalbumin (SeAlb), selenite and selenate in human serum using species-unspecific isotope dilution (SUID). The 3D chromatographic separation is proposed to remove typical spectral interferences in this matrix from chloride and bromide on (77)Se ((40)Ar(37)Cl), (80)Se ((79)Br(1)H) and (82)Se ((81)Br(1)H). In addition, a previous method based on 2D/SE-AF-HPLC is proposed as a simple alternative when low molecular mass selenium species are absent in the samples. The method is robust, reliable and fast with typical chromatographic runtime less than 35min. Detection limits are in the range of 0.2-1.3ng of Seg(-1). Method accuracy for determination of total protein-bound to Se was assessed by analyzing an human serum reference material (BCR-637) certified for total Se content and method reliability checked in samples of human serum providing results in good agreement with the total selenium concentration. In addition, the application of the method to commercial human serum and plasma reference materials for quality control analysis, certified for total Se, has provided, for the first time, indicative levels of selenium containing proteins in these samples.


Column-switching; Human serum; Inductively coupled plasma-mass spectrometer; Isotopic dilution analysis; Selenometabolites; Selenoproteins

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