High-performance liquid chromatography of selenium compounds utilizing perfluorinated carboxylic acid ion-pairing agents and inductively coupled plasma and electrospray ionization mass spectrometric detection

Increasing speciation demands in clinical chemistry, toxicology and nutrition have made the determination of the total elements in a sample inadequate; the amount of an element and the chemical forms in which it is present need to be known. Inductively coupled plasma mass spectrometry (ICP-MS) was used after high-performance liquid chromatographic (HPLC) separation, as was electrospray ionization mass spectrometry (ESI-MS). The effect of variation of the number of carbon atoms in perfluorinated carboxylic acids used as ion-pairing agents for the separation of selenium compounds was examined. Trifluoroacetic acid (0.1%), pentafluoropropanoic acid (0.1%) or heptafluorobutanoic acid (0.1%; HFBA) were alternatively used as additives to methanol–water (1:99, v/v) solutions as mobile phases. Reversed-phase HPLC–ICP-MS with 0.1% HFBA in the mobile phase allowed more than 20 selenium compounds to be separated in 70 min in an isocratic elution mode; the separation of natural selenium-enriched sample extracts was examined and explained. The pH of the 0.1% HFBA solution was modified with hydrochloric acid or ammonia and the pH of the sample extracts before injection was modified in order to overcome unwanted double peak formation in the chromatograms of sample extracts. Oxidations of standard γ-glutamyl-Se-methylselenocysteine and Se-methylselenocysteine were carried out using 30% H2O2 solution and identifications of selenium-containing oxidation products were made using HPLC–ICP-MS and HPLC–ESI-MS. The principal organic oxidation product in both cases was methaneseleninic acid (MeSeO2H).