Selenium species in aqueous extracts of alfalfa sprouts by two-dimensional liquid chromatography coupled to inductively coupled plasma mass spectrometry and electrospray mass Spectrometry detection

The complementary use of two different liquid chromatographic mechanisms coupled to inductively coupled plasma mass spectrometry (ICP-MS) for selenium ( Se) specific detection has permitted the screening of the most abundant Se-containing fractions in selenized alfalfa sprouts ( Medicago sativa). Aqueous extracts of the sprouts were fractionated first by size exclusion chromatography ( SEC) using a Superdex Peptide column and a mobile phase containing an ammonium acetate buffer (pH 7). Further purification of the individual SEC Se-containing fractions was carried out using two different chromatographic systems: a Shodex Ashaipack column, with a mixed mechanism of size exclusion and ion exchange, and a conventional reversed phase C-8 using ion-pairing reagents. In both cases, the columns were coupled to an inductively coupled plasma mass spectrometer equipped with an octapole reaction system for Se specific detection. This system allowed the on-line monitoring of the most abundant Se isotopes (Se-78, Se-80) by reducing the possible polytomic interferences affecting these ions by adding hydrogen (2 mL min(-1)) to the octapole reaction cell. The results obtained by both separation mechanisms were highly comparable, revealing the presence of Se-methionine and Se-methyl selenocysteine. Both compounds were then confirmed by analyzing the corresponding fractions by electrospray quadrupole-time-of-flight (ESI-Q-TOF) mass spectrometry. Finally, an additional Se-containing species showing Se isotope distribution was detected at a molecular ion m/z 239 in the ESI-Q-TOF. The collision-induced dissociation of the m/z 239 and 237 ions ( corresponding to Se-80 and Se-78 isotopes, respectively) revealed the possible presence as well of a derivative of the Se-2-propenyl selenocysteine.