Determination of Selenomethionine and Selenocysteine in Human Serum Using Speciated Isotope Dilution-Capillary HPLC−Inductively Coupled Plasma Collision Cell Mass Spectrometry

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Abstract
A method for the accurate determination of selenoamino acids in human serum by HPLC−ICPMS was developed using the species-specific isotope dilution analysis principle. A serum sample was enzymatically digested with a mixture of lipase and protease after derivatization of the selenocysteine residues with iodoacetamide. The selenoamino acid fraction was isolated by size exclusion LC followed by the separation of selenomethionine and the carboxymethylated selenocysteine by capillary HPLC. The isotope-specific determination of 77Se and 80Se was achieved on-line by ICP collision cell MS allowing the removal of polyatomic interferences. Quantification was carried out by isotope dilution using a 77Se-labeled selenomethionine spike and the determination of the 77Se/80Se ratio in the cHPLC selenomethionine peak. The accurately determined selenomethionine was used as an internal standard for the selenocysteine determination from the same chromatogram. The modification of the previously developed cHPLC−ICPMS interface allowed the reduction of the absolute detection limits twice (down to the 75-fg level), which resulted in the lowest ever reported procedural detection limits (below 0.5 ng g-1 for a 450-mg serum sample). The precision was less than 5% RSD. The method was validated by the mass balance of selenium (amino acid incorporated vs total).