Abstract

The potential of anion-exchange chromatography coupled to double focusing ICP-MS via a post-column isotope dilution analysis mode for the quantitative speciation of Fe, Cu and Zn in human serum is described. Protein separation was achieved with an anion-exchange column (Mono-Q HR 5/5) while the mobile phase consisted of a gradient of initial buffer solution (0.05 M Tris-HCl, pH = 7.4) and the final buffer solution (0.5 M ammonium acetate in 0.05 M Tris-HCl, pH = 7.4). After protein separation, a mixed solution containing the enriched isotopes, ⁵⁷Fe, ⁶⁵Cu and ⁶⁷Zn, was continuously pumped and mixed with the biological species eluting from the column. The analytical signals for Fe (⁵⁶Fe/⁵⁷Fe), Cu (⁶³Cu/⁶⁵Cu) and Zn (⁶⁴Zn/⁶⁷Zn) were monitored on a double-focusing inductively coupled plasma mass spectrometry (ICP-MS) instrument operated at medium resolving power (m/Δm = 3000). This resolution allowed adequate separation of polyatomic interferences affecting the main isotopes of Fe, Cu and Zn in serum. The conventional abundance vs. time chromatogram obtained was converted to a “mass flow chromatogram” and the amount of metal in the different fractions (biomolecules) was obtained by integration of such chromatographic peaks. Total metal contents of Fe, Cu and Zn were also determined by direct isotope dilution analysis (IDA) of the serum samples, diluted 1∶20, using the same ICP-MS. The method proposed was finally applied to the speciation of serum samples from healthy volunteers and patients on hemodialysis. Some speciation differences were observed between both populations, particularly for Fe speciation, indicating that quantitative speciation studies could be useful for disease diagnostic purposes.