HPLC-AAS hybrid technique for studying the speciation of trace metals (Al, Fe, Si, Hg) in biological fluids: A review of applications, recent experiences and perspectives

A method developed using the combination of HPLC and AAS enabling both qualitative and quantitative study of the protein binding and speciation of trace metals in biological fluids at clinical relevant levels is reviewed. The whole system was made metal-free by using polymer-based columns, column holders and tubing and by the insertion of a silica-based scavenger column placed immediately before the injection valve to selectively retain any trace of aluminum and iron originating from buffer solutions and recipients. ETAAS instrumental conditions were carefully selected to eliminate interferences secondary to the salt gradient elution. Particular attention was paid to the choice of HPLC columns. Protein recoveries varied between 95 and 105% and trace metal recoveries were close to 100%. Intra-assay and inter-assay CVs of the HPLC/AAS hybrid technique were below 10%. Because of its high sensitivity, the method can be used at clinically relevant concentrations and was applied successfully to study (i) the interaction between iron and aluminum for binding to transferrin, (ii) the influence of citrate on the transferrin binding of aluminum, (iii) the speciation of silicon in the serum of dialysis patients and (iv) the toxicity of mercury compounds in cell cultures.