Abstract

This work describes the separation of the enantiomeric forms of Se-amino acids by reversed phase liquid chromatography following formation of the diastereomers using 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (Marfey's reagent) as the derivatizing agent. Optimization of the chromatographic conditions by UV absorption at 340 nm (λ_max for Marfey's derivatives) was possible. The final operating conditions involved the use of 50% (v/v) MeOH at pH 5.3 (acetic acid–sodium acetate), which provided a good separation of the diastereomers of Se-methionine within 26 min. Slight modification of the sample introduction system by on-line dilution (1∶4) of the chromatographic eluent using a PTFE tee piece was required for good performance of the ICP-MS. Final instrumental set-up involved the use of both detectors on-line (diode array and ICP-MS). Under these conditions, detection limits at the ppb level (11 ppb as Se in the Se-methionine diastereomer) using ICP-MS detection were obtained with negligible band broadening caused by the on-line dilution. The methodology was finally applied to the qualitative identification of D and L enantiomers in selenium nutritional supplements (selenized yeast and tablets) and a pharmaceutical preparation.