Protein fractionation and Cd-speciation in human breast milk by HPLC and voltammetry

A method for preparative separation of proteins from human breast milk was developed, which preserved the intactness of metal-protein complexes as well as keeping the sample injection and fraction harvesting free of Cd-contamination. Preliminary, incomplete separation was performed on a TSK 60 x 4 mm ID column, in order to check the retention time of the Cd-protein complex in relation to the elution times of protein standards. The Cd-profiles were determined by DPASV (differential pulse anodic stripping voltammetry) and showed concentrations below 100 ng/L (detection limit) except in the fraction 55-59 min, where about 1 microgram/L of Cd in human milk was determined. This Cd-elution time was in accordance with the retention of the metallothionein (Mt) standard (57 min) of the above mentioned column. Further attempts to identify the protein nature of the Cd-species were carried out by isoelectric focusing. The "Mt-fraction" of breast milk was focused and the resulting distribution profile compared to the profile of the Mt-standard. Good correspondence was achieved. Further investigation will be based on the use of a more efficient column (TSK 1000 x 8 mm ID) for separation and further identification of the Cd-species.