Determination of Cu, Fe, Mn, and Zn in blood fractions by SEC-HPLC-ICP-AEScoupling

The binding of Cu, Fe, Mn, and Zn to proteins in blood and in blood fractio ns was investigated, since their interactions in free radical metabolism in humans is of great interest. An HPLC-ICP-AES technique was developed allow ing adequate separation of metalloproteins and of inorganic and organic met al species. For the separation of metalloproteins in erythrocytes and blood plasma a Merck Superformance Fractogel EMD BioSEC 650 (S) column was used. Size exclusion chromatography (SEC)-HPLC was hyphenated to ICP-AES both on -line and off-line for the detection of trace elements in the fractions res ulting from HPLC separations. HPLC parameters, pH, temperature, flow rate a nd salt concentration were optimized for the protein separation and the opt imal conditions were applied for the hyphenation to the ICP-AES detector. T he separation column was calibrated with five standard proteins. For the el ement determination by ICP-AES a line selection with respect to the sensiti vity was performed. Three different methods were used for the determination of trace elements in blood: direct determinations, on-line and off-line SE C-HPLC-ICP-AES measurements. For the optimizing experiments blood samples o f one female subject were used. The direct determination by ICP-AES of the elements was performed in blood and blood fractions of ten different subjec ts to obtain the average concentration ranges; From the results the identif ication of the protein Cu/Zn superoxide dismutase in erythrocytes was possi ble. The LOD were 0.03 mu g mL(-1) for Cu, 0.026 mu g mL(-1) for Fe, 0.8 ng mL(-1) for Mn, and 0.09 mu g mL(-1) far Zn in a synthetic blood matrix.