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 ⁷⁷Se (⁴⁰Ar³⁷Cl) and ⁸²Se (⁸¹Br¹H). 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. Figure A methodology for the alleviation of Cl and Br interferences in the accurate simultaneous speciation analysis of glutathione peroxidase, selenoprotein P and selenoalbumin in human serum by affinity HPLC coupled on-line with ICP-quadrupole MS is proposed. This approach may be particularly useful for clinical laboratories that only have an ICP-quadrupole MS without a collision cell, or that lack an expensive ICP-SFMS (high-resolution) instrument