Speciation analysis of selenoproteins in human serum by solid-phase extraction and affinity HPLC hyphenated to ICP-quadrupole MS

Anion exchange-solid-phase extraction carried out prior to double column affinity-high performance liquid chromatography (AF-HPLC) coupled on-line with conventional inductively coupled plasma-(quadrupole) mass spectrometry is proposed for simultaneous speciation analysis of selenoprotein P (SelP), glutathione peroxidase (GPx) and selenoalbumin (SeAlb) in human serum. Efficient inteference-free free AF-HPLC separation of GPx, SelP and SeAlb is obtained within a total chromatographic run time less than 15 min. The repeatability in terms of relative standard deviation (R.S.D., %, n = 10) is 7% for GPx and SelP and 4% for SeAlb. The detection limits are 0.1 ng mL(-1) for GPx, 1.0 ng mL(-1) for SelP and 1.3 ng mL(-1) for SeAlb. The method was validated by the analysis of two human serum reference materials certified for total Se content.