Quantitative determination of iron citrate/malate complexes by isotope–dilution hydrophilic interaction liquid chromatography – electrospray MS/inductively coupled plasma MS

Abstract

Iron occurs in plant fluids principally in the form of various low molecular species of which the quantitative determination is hampered by the dynamic equilibria and unavailability of authentic standards. The high-performance liquid chromatography (HPLC) separation of these species is affected by the lability of the complexes and reactivity with the column stationary phase, the phenomena which further affect quantitative analyses. A quantification method based on the in situ formation of isotopically-labelled iron species reflecting their composition in the sample, and hence to be used as internal standards, was developed. The mixture of iron species with the natural and enriched composition was subsequently baseline separated by hydrophilic interaction ion chromatography (HILIC). Each of the species was quantified by either electrospray ionization mass spectrometry (ESI MS) or inductively coupled plasma mass spectrometry (ICP MS) by the comparison of the intensity of peaks corresponding to the molecules containing natural and isotopically-enriched iron. Prior knowledge of the molecular structures of the iron species is not required and hence the need for molecular standards is eliminated. The similar chemistry of isotopically labelled and non-labelled species allowed the correction of the on-column phenomena, such as species retention or degradation, formation of artefacts, or change in sensitivity because of the changing composition of the gradient. The applicability of the method was demonstrated for the quantification of mixed-ligand multicore iron complexes with malate and citrate in coconut water used as a model endosperm liquid.