Selenium speciation in human urine samples by LC- and CE- ICP-MS—separation and identification of selenosugars Bente Gammelgaard* and Lars Bendahl Department of Analytical Chemistry, The Danish University of Pharmaceutical Sciences, Universitetsparken 2, DK-2100 Copenhagen, Denmark. E-mail: bg@dfh.dk; Fax: 145 3530 6010 Received 3rd July 2003, Accepted 3rd September 2003 First published as an Advance Article on the web 26th September 2003 Human urine samples were analysed by a reversed-phase chromatographic system and an ion-pair chromatographic system. The chromatographic system was connected to the ICP-MS either by a microconcentric nebulizer (MCN) in combination with a cyclonic spraychamber or by a modified direct injection nebulizer (MDIN). The sensitivity of the latter was better than the sensitivity of the MCN, which on the other hand was more robust for the analysis of samples with high concentrations of dissolved solids. Urine sample composition did not seem to change when urine was exposed to evaporation under nitrogen at ambient temperature and methanol extraction. A pre-concentration factor of 10 was achieved with this procedure. On occasions when a pre-concentration factor of 100 was obtained by lyophilsation and methanol extraction, at least 10 selenium compounds were separated in the urine sample. Urine samples were collected from two healthy volunteers who had been supplied with 1000 mg and 2000 mg of selenium, respectively, in the form of selenized yeast. When samples were spiked with 8 different standards, only two standards co-eluted with compounds in urine in both chromatographic systems: the major urinary metabolite Se-methyl-N- acetylgalactosamine and Se-methyl-N-acetylglucosamine. The presence of Se-methyl-N-acetylglucosamine in urine was verified by co-migration with the standard in capillary electrophoresis after fractionation by preparative reversed-phase chromatography. Se-methyl-N-acetylglucosamine is only a minor metabolite as its concentration was less than 2% of the concentration of Se-methyl-N-acetylgalactosamine. The presence of this metabolite in urine has, to our knowledge, not been suggested before. Trimethylselenonium, selenomethionine, Se-methylselenocysteine, Se-methylselenomethionine and selenocystamine were not detected in these samples. Introduction The essential trace mineral selenium is indispensable for the functioning of the human body. Selenium exerts its effects via the selenoproteins that often participate in anti-oxidative or catalytic processes. A review on selenium-containing natural products, including selenoproteins, has recently been given. 1 According to this, 18 mammalian selenoproteins have now been defined by sequence. Selenium deficiency causes various adverse health effects 2 and in recent years the element has gained increasing interest owing to its possible cancer protective effects. 3 Results of experiments on the cancer protective effect of monomethylated selenium compounds such as selenobetaine, Se-methylseleno- cysteine and methylseleninic acid suggest that formation of a monomethylated selenium metabolite is important for cancer chemoprevention. 4–6 Selenium metabolism The most often presented model on the metabolism of selenium is primarily based on experiments on rats after administration of large doses of selenium. 7,8 According to this model, seleno- methionine is either incorporated unspecifically into proteins in competition with methionine or is transformed via selenocys- teine to selenide, from which it enters the selenoproteins after synthesis to selenocysteine. Excess selenium is excreted via methylation to monomethyl selenol, dimethylselenide, which is exhaled via the breath, and to trimethylselenonium which is excreted in urine. This pathway is shown in Fig. 1 as the pathway for toxic levels. The metabolism of inorganic selenium in sub-toxic doses in rats has been thoroughly described by Suzuki and co-workers on the basis of administration of 82 Se-enriched selenite and selenate and subsequent determination of the 82 Se level in organs and body fluids by size exclusion chromatography and ICP-MS detection. 9 Intravenously injected selenite was selectively taken up by the red blood cells (RBC) 10 and reduced by glutathion (GSH) to selenide. Selenide was transported to the liver, where it was incorporated into selenoprotein P, re-excreted to the bloodstream and transferred to the kidneys, where it was degraded and used for synthesis of extra-cellular glutathion peroxidase. 9 Contrary to selenite, selenate was not taken up by RBCs but was retained in the bloodstream until it entered the liver or was excreted directly in urine. The metabolic products of selenite and selenate were not distinguishable. 11 The selenium in Fig. 1 Model of selenium metabolism modified from the original model by Ip 5 and supplemented with the latest results of the work from the group of Suzuki. 13 GS-: Glutathion conjugate. DOI: 10.1039/b307539g J. Anal. At. Spectrom. , 2004, 19 , 135–142 135 This journal is ß The Royal Society of Chemistry 2004