Simultaneous speciation analysis of glutathione peroxidase, selenoprotein P and selenoalbumin in human serum by tandem anion exchange-affinity HPLC and on-line isotope dilution ICP-quadrupole MS

A method based on anion exchange (AE) and affinity (AF)-HPLC(AE-AF- HPLC) hyphenated to inductively coupled plasma-(quadrupole) mass spectrometry (ICP- QMS) was developed for the speciation analysis of selenoprotein P (SelP), glutathione peroxidase (GPx) and selenoalbumin (SeAlb) in human serum. AE- HPLC is proposed here for the on- line alleviation of Cl and Br spectral interferences on (77)Se ((40)Ar(37)Cl) and (82)Se ((81)Br(1)H). Separation of GPx, SelP and SeAlb by AE-AF- HPLC was obtained within a total chromatographic runtime of < 20 min. On-line (post-column) isotope dilution (ON-ID) and on- line external calibration (ON-EC)ICP- QMS were used for the quantification of Se in GPx, SelP and SeAlb. ON-EC using a Se-L-cystine standard was shown to be a suitable approach for the routine simultaneous speciation analysis of serum GPx, SelP and SeAlb. The method validation was carried out by direct ICP-sector field MS determination of Se in GPx, SelP and SeAlb fractions collected after AE-AF-HPLC separation. In addition, the method accuracy for the determination of total protein-bound Se was assessed by analyzing a human serum reference material (BCR-637) certified for total Se content.