Study of selenium species distribution in biological tissues by size exclusion and ion exchange chromatagraphy inductively coupled plasma–mass spectrometry

Selenium species present in water-soluble fractions of proteins of lyophilised biological samples, such as fish (tuna and trout), shellfish (krill, oyster and mussel), vegetal tissues (white clover and wheat flour) and selenized yeast were evaluated. The water-soluble fractions with and without 4% (w/v) sodium dodecyl sulphate were analysed by size exclusion chromatography coupled to both inductively coupled plasma–mass spectrometry (SEC–ICP–MS) and UV spectrophotometers. Two different columns with effective separation ranges between 300 and 1 kDa (Biosep-SEC-2000) and 7 and 0.1 kDa (Superdex Peptide HR 10/30) were used, with 5 mM sodium phosphate at pH 7.2 (0.8 ml min^-1) and 5 mM sodium phosphate with 60 mM NaCl at pH 7.5 (0.6 ml min^-1) as mobile phases, respectively. The enzymatic digests of both the water-soluble fractions and the non-soluble residues were analysed by cation exchange chromatography–ICP–MS, using a Hamilton PRP-X200 column and 4 mM pyridine (1 ml min^-1) at pH 2.8 and 4.7 as mobile phases. The presence of high molecular weight selenoproteins (150–50 kDa) in the samples studied, except for fish and white clover with proteins <30 kDa, was demonstrated and low molecular weight (<5 kDa) compounds were also found. Selenomethionine species (SeMet) within the range of 0.2–600 (mu)g g^-1 was quantified in the selected tissues, although selenocystine (SeCys2) was identified only in vegetal samples (0.1–0.7 (mu)g g^-1). Trimethylselenonium ion was quantified (0.1–0.3 (mu)g g^-1) in oyster, mussel, trout and white clover tissues. Inorganic selenium as selenite was found in the selenized yeast, krill, and white clover samples (0.2–3 (mu)g g^-1), while selenate was found to be present only in yeast tissues (8 (mu)g g^-1).