Selenium speciation in pretreated human urine by ion-exchange chromatography and ICP-MS detection

Urine samples were extracted by benzo-15-crown-5-ether to remove sodium and potassium. More than 90% of the sodium and potassium content of the urine was removed with this extraction. In a cation-exchange system based on oxalic acid at pH 3, chromatography of an untreated urine pool resulted in a large peak in the front together with three small peaks. In the crown ether treated pool at least five signals were obtained. When the eluent was ammonium formate at pH 3, two small signals together with a large signal in the front were obtained in untreated urine, while three more distinct peaks and a peak in front were obtained in the crown ether extracted urine. In both systems, two of the peaks co-eluted with selenomethionine (SeMet) and the trimethylselenonium ion (TMSe). None of the signals co-eluted with either selenocystine or selenoethionine. Urine samples from different individuals showed different concentrations and ratios of the selenium species present. There was no difference in the chromatograms when the urine pool was treated with alpha -glucuronidase or ultrafiltrated through a membrane with a cut-off value of 10 kDa. When the urine pool was analysed by capillary electrophoresis ICP-MS at pH 8.2, four peaks could be separated. One of the peaks co-migrated with SeMet while TMSe did not appear in this system. Crown ether extraction did not improve the separation. Hence, apart from SeMet and TMSe, at least three more unknown selenium-containing species were present in urine.