Copper fractionation by SEC-HPLC and ETAAS: Study of breast milk and infant formulae whey used in lactation of full-term newborn infants
This method will allow the determination of bound copper to low relative molecular mass compounds in milk. The milk whey obtained by ultracentrifugation was submitted to fractionation by size exclusion chromatography (SEC) on a TSK-Gel2000 (Toso Haas) column with a mobile phase of 0.2 M NH4NO3 + NH3, pH 6.7. Fractions of effluent corresponding to the protein peaks were collected and the copper content was determined by ETAAS. The method was sensitive (LOD 0.4 μg l−1 and LOQ 1.5 μg l−1 in the fraction; LOQ 7.5–22.5 μg l−1 referred to the milk sample and depended on fraction volume) and precise (RSD ± 10%). Media sample recoveries from the column were 101.2%. Cu was predominantly present in fractions corresponding to relative molecular mass 76 and 15 kDa of breast milk while copper was mostly found in fractions corresponding to 14 and 38 kDa of cow’s milk-based infant formulae; moreover, copper was eluted in the relative molecular mass region <6 kDa.