Determination of the total content and binding pattern of elements in human milk by high performance liquid chromatography-inductively coupled plasma atomic emission spectrometry

Evidence of element speciation in biological fluids is scarce and fragmentary in the literature. On the other hand, it is well known that chemical speciation analysis is prone to error due to contamination, modification or breaking up of the metal–organic moiety link during the separation processes, as well as to insufficient detection power for element quantification in the various fractions. Therefore, there is a keen need for sensitive and reliable speciation methods. We explored the analytical capabilities of size exclusion chromatography (SEC) in conjunction with ultrasonic nebulization inductively coupled plasma atomic emission spectrometry (UN-ICP-AES) for the speciation of Ca, Cu, Fe, Mg, Mn and Zn in human milk. We applied the method to assess concentration range and binding pattern of the said elements in the milk of 60 lactating mothers (19–40 years of age) representative of different areas of Italy. The total concentration ranges found were in good agreement with those reported in the literature. These levels were as follows (in ng ml−1): Ca, 306 000±11 760; Cu, 370±30; Fe, 650±40; Mg, 23 000±510; Mn, 30±2; and Zn, 2720±70. The influence of factors such as age (over or under 30) and residence area (urban or rural) of the mothers on the total concentration of the elements was also evaluated. Magnesium concentration was higher for mothers >30, while higher Zn levels were found for maternal age <30. Copper was higher in women living in urban areas. Moreover, strong positive Ca–Mg and Fe–Mn associations and significant negative Cu–Zn correlations were found. The chromatographic separation yielded five fractions. The first of these presumably accounted for caseins (αs, β and k) aggregates; the second peak mainly contained immunoglobulins (Igs); the third could be traced back to human serum albumin (HSA) and lactoferrin (LF); the fourth could be entirely ascribed to α-lactalbumin (α-La); and the fifth accounted for all substances with relatively low molecular weights, such as non-proteic compounds. As anticipated, the elements investigated showed peculiar associations with the components of the milk. Copper and Fe appeared homogeneously spread over all the organic components. Calcium and Mg preferably bound to low molecular weight compounds. Manganese had a peculiar pattern as it was found in considerable percentage in the first and fifth fractions. As for Zn, the highest percentage was found in the fraction corresponding to α-lactalbumin.