Reversed-phase liquid chromatography with mixed ion-pair reagents coupled with ICP-MS for the direct speciation analysis of selenium compounds in human urine

A sensitive and robust method for the determination of five inorganic and organic Se species in human urine by reversed-phase liquid chromatography with mixed ion-pair reagents coupled with inductively coupled plasma mass spectrometry (HPLC-ICP-MS) is described in this paper. A good separation for anionic, cationic and neutral Se species, namely selenate [Se(VI)], selenourea (SeUr), selenomethionine (SeMet), selenoethionine (SeEt), and trimethylselenonium ion (TMSe), was achieved within 15 min on a LiChrosorb RP 18 reversedphase column by using mixed ion-pair reagents of 2.5 mM sodium 1-butanesulfonate and 8 mM tetramethylammonium hydroxide, with isocratic elution at a flow rate of 1.0 mL min. The detection limits of the five Se species obtained by HPLC-ICP-MS ranged from 0.6 to 1.5 ng Se mL using an injection volume of 20 mL. It is noteworthy that the urine sample can be directly injected into the analytical system without any pre-treatment, except a filtration with a 0.45 mm membrane filter, and that the determination of Se species was free from chloride-induced matrix interference. In addition, no serious deterioration in column performance or decrease in the sensitivity of ICP-MS was observed for the experimental period of three months. In Japanese urine samples, no detectable SeMet, SeUr, and SeEt could be found, even if the total Se concentration was higher than 100 ng Se mL. On the contrary, TMSe and two unknown Se species (U1 and U2) were detected in the urine. The major unknown, U1, was found in all of the measured urine samples, suggesting that it might be one of the important Se metabolites.