QUANTITATIVE HPLC ETAAS HYBRID METHOD WITH AN ONLINE METAL SCAVENGER FOR STUDYING THE PROTEIN-BINDING AND SPECIATION OF ALUMINUM AND IRON/

A quantitative metal-free high-performance liquid chromato-graphic/ele ctrothermal atomic absorption spectrometric (HPLC/ETAAS) technique all owing an adequate separation of the proteins and of the inorganic/orga nic metal species of interest was developed. A silica-based scavenger was placed proximal to the injection valve retaining any remaining Al and Fe originating from buffer solutions and recipients. ETAAS instrum ental conditions and matrix modifiers were carefully selected to elimi nate interferences occurring due to the salt gradient elution. The pos telution Al recovery (143-1428 mu g L(-1); 100-mu L sample volume) was found to be 100.1 +/- 15.3% (N = 50) whereas that of Fe was 97.3 +/- 2.0% (N = 20; 442-2653 mu g L(-1)). The protein recovery varied betwee n 95 and 105%. The HPLC/ETAAS intraassay CV (N= 3) was 8.0% for Al (el uted amount of Al bound to transferrin (Tf), 7.3 ng) whereas that of F e was 1.4% (eluted amount of Fe bound to Tf, 124.8 ng). The intraassay CV of the HPLC Tf determination was 1.6%. The proposed method is sens itive and accurate, allowing for the first time quantitative evaluatio n of the competition between Al and Fe for protein binding at clinical relevant concentrations. The technique will also be used for studying the protein binding of elements other than Al and Fe.