Advanced quality control for zinc speciation investigations in human breast milk

Information is provided about the reliability of results obtained in Zn-speciation analyses of human milk. For such analyses, convenient techniques have already been given in an earlier paper [1], comprising mass balances of metal and ligand, purity of analytes, etc. Here, however, the influence of the eluent was checked in an extended manner. For this series of experiments, bi-distilled water was compared with different buffers as the eluent. Water proved to be a more suitable mobile phase than buffers with regard to contamination. Moreover, a method was developed to investigate the possibility of a transfer of Zn among the proteins during size exclusion chromatography. For these experiments, casein and metallothionein were chosen as competitive Zn-ligands, showing extremely different affinities to Zn. A possible Zn-transfer from one protein to the other was examined in combining a Zn-containing protein with a Zn-free one. No change of the Zn-status of the proteins was detected, indicating a stable protein-metal complex under the experimental conditions. Zn and protein mass balances (injected/eluted) were calculated and found to be 100%.