IntroductionSelenium (Se) is an essential trace element at low levels of intake and produces toxic symptoms when it is ingested at levels higher than those required for adequate nutrition.1Selenium deficiency has been associated with several diseases,e.g., heart diseases and cancer.2Recently, it was reported that sufficient selenium supplement can protect against cancer.3A variety of Se-enriched materials, such as garlic, yeast, and lactic acid bacteria, have been studied for the purpose of selenium supplementation. With the increased use of Se for dietary supplementation in animals and humans, the chemical forms and quantities of Se in urine are of interest because urinary excretion is the main route of Se elimination and more than 50% is excreted this way. Moreover, the urinary Se speciation may provide information about the Se status of the body, either as a detoxified formor as the metabolite of an essential chemical form.4To date, besides the inorganic Se compounds, the only organic Se compound identified conclusively in human urine has been TMSe+.5,6Evidence for the presence of selenourea (SeUr) has also been reported.7Selenoamino acids, such as selenomethione (SeMet) and selenocystine (SeCys) have been hypothesized as metabolites in the biological pathways of Se.8,9Due to the complex urine matrix and low concentration of Se (normally less than 100 ng mL−1), analytical methods for urinary speciation of Se are very scarce. Earlier methods often required a large volume of urine samples (1–2 l) and employed a series of chemical pretreatment steps, such as precipitation/coprecipitation,10thermal decomposition and derivatization of selenoamino acids.11,12Such sample treatments may have changed the original Se species present in urine, thus resulting in erroneous results. In recent years, the development of analytical chemistry methods based on the hyphenation of HPLC with ICP-MS have been reported in the literature. In order to separate Se species, different separation modes, such as ion-exchange,4,13–15reversed-phase,16–19ion-pair reversed-phase20and vesicle-mediated chromatography,19,21have been examined. Some difficulties, however, have been encountered when the different HPLC methods were applied to urine samples,e.g., the inability to separate inorganic Se species together with organic ones. Some reported methods can only separate two or three Se compounds, either inorganic or organic species, and, in some methods, the conclusively identified metabolite TMSe+was not considered at all in the separation system.18,19,21On the other hand, many reported methods are not robust enough to maintain their separation performance in urine samples. Furthermore, due to the lack of sensitivity, some methods can only work with spiked urine samples.21,22In order to minimize urine matrix interference in both the chromatographic separationand the ICP-MS detection steps, solid-phase extraction,18ethanolic precipitation5and dilution14–16,19,20,23sample pretreatment steps have been carried out. However, there is a strong chance that these sample preparations alter the composition of the original sample24or cause loss of the analytes of interest. Therefore, analytical methods that are sensitive and highly robust for direct urinary Se speciation analysis are urgently required.In this paper, we report a sensitive and robust HPLC-ICP-MS method for urinary Se speciation analysis. This method was developed to meet the demand of our ongoing research project on the study of Se metabolism of Se–yeast supplements. By using a unique reversed-phase chromatographic separation system with mixed ion-pair reagents,25,26in which both anionic (sodium 1-butanesulfonate) and cationic (tetramethylammonium hydroxide) ion-pairing reagents were added simultaneously into an aqueous solution, five Se species, Se(vi), SeUr, TMSe+, SeMet, SeCM, and SeEt, could be directly analyzed in human urine samples. Due to its remarkable analytical characteristics, such as low detection limits and highly matrix tolerance, this method provides a powerful tool for the study of Se metabolism and for the dose control of ingested Se nutritional supplements.