In human milk zinc (Zn) is bound to proteins and low molecular mass (LMM) ligands. Numerous
investigations demonstrated that Zn bioavailability in human milk is for infant much higher than in
cow’s milk. It was presumed that in the LMM human milk fraction highly bioavailable Zn-citrate
prevails. However, literature data are controversial regarding the amount of Zn-citrate in human milk
since analytical procedures reported were not quantitative. So, complex investigation was carried out
to develop analytical method for quantitative determination of this biologically important molecule.
Studies were performed within the pH range 5–7 by the use of synthetic solutions of Zn-citrate
prepared in HEPES, MOPS and MES buffers. Zn-citrate was separated on weak anion-exchange
convective interaction media (CIM) diethylaminoethyl (DEAE) monolithic chromatographic column
using NH4NO3 as an eluent. Separated Zn species were determined by flame atomic absorption
spectrometry (FAAS) or inductively coupled plasma mass spectrometry (ICP-MS). Quantitative separation
of Zn-citrate complexes ([Zn(Cit)] and [Zn(Cit)2]
4; column recoveries 94–102%) and good
repeatability and reproducibility of results with relative standard deviation (RSD73.0%) were obtained.
In fractions under the chromatographic peaks Zn-binding ligand was identified by electrospray
ionization tandem mass spectrometry (ESI-MS-MS). Limits of detection (LOD) for determination of
Zn-citrate species by CIM DEAE-FAAS and CIM DEAE-ICP-MS were 0.01 mg Zn mL1 and 0.0005 mg
Zn mL1
, respectively. Both techniques were sensitive enough for quantification of Zn-citrate in human
milk. Results demonstrated that about 23% of total Zn was present in the LMM milk fraction and that
LMM-Zn corresponded to Zn-citrate. The developed speciation method represents a reliable analytical
tool for investigation of the percentage and the amount of Zn-citrate in human milk.
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