Quantitative selenium speciation in human urine by using liquid chromatography–electrospray tandem mass spectrometry

A liquid chromatography–electrospray-tandem mass spectrometry (ES-MS/MS) method was developed for the speciation analysis of four organic selenium species of relevance to human urinary metabolism, namely trimethylselenomium ion (TMSe+), selenomethionine (SeMet) and the two selenosugars, methyl 2-acetamido-2-deoxy-1-seleno-β-d-galactos/-glucos-amine (SeGalNAc and SeGluNAc, respectively). Their chromatographic separation was achieved by using a cation exchange pre-column coupled in-series with a reversed-phase high-performance liquid chromatography column, along with an isocratic mobile phase. Online detection was performed using ES-MS/MS in selective reaction monitoring mode. SeGalNAc was detected as the major human urinary metabolite of selenium in the samples analysed, whereas TMSe+ was detected in the urine of one volunteer before and after receiving a selenium supplement. SeMet was not detected as a urine excretory metabolite in this study. Spiking experiments performed with the urine samples revealed significant signal suppression caused by coeluting matrix constituents. To overcome such interferences, isotopically labelled 13CD382SeGalNAc was used as an internal standard, whereas in the absence of an isotopically labelled internal standard for TMSe+, the standard addition method was applied. Quality control for the accurate quantitation of TMSe+ and SeGalNAc was carried out by analysing spiked human urine samples with appropriate selenium standards over a concentration range of 10–50 μg Se L−1. The method has achieved a limit of detection in the presence of urine matrix comparable to that of HPLC-inductively coupled plasma-mass spectrometry for the four selenium species: 1.0 μg Se L−1 for TMSe+, 5.6 μg Se L−1 for SeMet, and 0.1 μg Se L−1 for both SeGalNAc and SeGluNAc.

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► Development of a selected reaction monitoring mass spectrometric method for the identification of Se species in human urine.
► A selenosugar was detected as the major human urinary metabolite of selenium in the samples analysed.
► The trimethylselenonium ion was detected in the urine of one volunteer before and after receiving a selenium supplement.
► Strict quality control measures were applied to validate identification.
► Quantitation was conducted using an isotopically labelled internal standard and the standard additions methodology.