Speciation analysis of selenoproteins in human serum by microbore affinity-HPLC hyphenated to ICP-Sector field-MS using a high efficiency sample introduction system
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Microbore affinity-HPLC (AF-HPLC) hyphenated to inductively coupled plasma-sector field mass spectrometry (ICP-SFMS) was applied to the simultaneous speciation analysis of glutathione peroxidase (GPx), selenoprotein P (SelP) and seleno-albumin (SeAlb) in 5 μL of (human) serum. ICP-SFMS (high resolution mode, HR, M/ΔM = 10,000) was used for the quantification of selenium in GPx, SelP and SeAlb after their separation by AF-HPLC, without prior sample preparation or mathematical correction of the spectral interferences. In order to compensate for the loss in sensitivity due to the detection in HR mode and the very low amount of sample taken for analysis, a high-efficiency sample introduction system was used as an interface between AF-HPLC and ICP-SFMS. This led to a signal-to-noise ratio enhancement of about one order of magnitude compared to the conventional experimental ICP-SFMS set-up. Apart from the very low sample consumption, another major advantage of the method described here is the significant reduction of the analysis time (≤7 min). Quantification of GPx, SelP and SeAlb was carried out using on-line (post column) isotope dilution; comparison with on-line external calibration by using Se-L-cystine standard is also addressed. The method accuracy for the determination of total protein bound Se was assessed by the analysis of a human serum reference material (BCR-637) certified for total Se content.
KeywordsSpeciation analysis Human serum selenoproteins Microbore affinity chromatography Inductively coupled plasma-sector field mass spectrometry On-line isotope dilution
This study is a contribution to the Marie-Curie Intra-European Project (MEIF-CT-2006-024156/ELSA-BIM) funded by the European Commission and to the iMERA-Plus project (TRACEBIOACTIVITY) funded by the European Metrology Research Programme (EMRP). The authors thank Drs. Warren Cairns and Clara Turetta (IDPA-CNR) for the scientific discussions. ELGA LabWater is acknowledged for providing the PURELAB Option-R and Ultra Analytic systems, which produced the ultra-pure water used in these experiments.
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