Anion exchange liquid chromatography–inductively coupled plasma-mass spectrometry detection of the Co 2+, Cu 2+, Fe 3+ and Ni 2+ complexes of mugineic and deoxymugineic acid

Abstract

Phytosiderophores, such as mugineic and deoxymugineic acid, are produced by graminaceous plant species in response to Fe deficiency conditions normally experienced in calcareous and alkaline non-calcareous soils. As these phytosiderophores have the ability to form thermodynamically stable complexes with other metal cations present in the growing medium, they have also been implicated in the transport and bioavailability of these metals in the environment. However, routine analytical methodology to detect the various metal complexes formed by these phytosiderophores is lacking. Therefore, as these complexes are negatively charged over a wide range of pH values, anion exchange liquid chromatography (AE LC) coupled to inductively coupled plasma-mass spectrometry (ICP-MS) was investigated as a means to separate and quantify these complexes. The metal–phytosiderophore complexes were prepared at pH 7 and separated by NaOH or NH 4NO 3 gradient elution on a Dionex AS11 anion exchange column. Of the metals tested only the Co 2+ and Ni 2+ complexes of mugineic and deoxymugineic acid were detected when using a 0–20 mM NaOH gradient elution profile. However, the phytosiderophore complexes of Cu 2+ and Fe 3+ were also detected when using NH 4NO 3 as the mobile phase at pH 7. Base-assisted hydrolysis of the latter two complexes is proposed to explain their apparent ‘instability’ in the high pH NaOH mobile phase. The absolute detection limits of the developed methodologies for these metal complexes ranged from 0.1 to 2.8 pmol. As phytosiderophore complexes with Cd 2+ and Zn 2+ were not detected, it was concluded that the dissociation kinetics of these metal–phytosiderophore complexes were too rapid for these complexes to be observed in the present chromatographic conditions.