Separation and identification of phytosiderophores and their metal complexes in plants by zwitterionic hydrophilic interaction liquid chromatography coupled to electrospray ionization mass spectrometry.

A sensitive method for the separation of different phytosiderophores (PS) of the mugineic acid (MA) family, and the candidate ligand for intracellular metal transport in plants nicotianamine (NA), and respective metal complexes in plants by zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) coupled to electrospray ionization mass spectrometry (ESI-MS) is described. Separation of mugineic acid, 2'-deoxymugineic acid (DMA), 3-epi-hydroxymugineic acid (epi-HMA), nicotianamine, Fe(III)-DMA, Fe(III)-NA, M(II)-DMA, and M(II)-NA complexes (M(II)=Zn(II), Cu(II), Ni(II), and Fe(II)), was achieved within 22 min on the ZIC-HILIC column by using a gradient elution with a mobile phase consisting of ammonium acetate and acetonitrile at pH 7.3, at a flow rate of 0.15 mL/min. The on-line coupling to ESI-MS in the negative ionization mode enables the detection of these compounds in the micromol/L range, which is the relevant concentration range in real plant samples. DMA-complexes of Fe(III), Zn, and Cu in wheat root, and an NA-complex of Ni in Arabidopsis were detected and identified by the proposed method. Even in the case of partial coelution of some divalent metal complexes, the identification is possible by their distinct mass spectra. The stability of metal complexes during separation was checked by injecting ethylenediaminetetraacetic acid (EDTA) after each run of metal-phytosiderophore complexes. Good stability of divalent-phytosiderophores, except for Fe(II)-complexes, was observed. During gradient separation, Fe(III)-complexes are partly dissociated (<20%), but a good sensitivity of Fe(III)-DMA in real plant samples is still achieved. In order to avoid instability problems with the separation of Fe-complexes, an isocratic separation is proposed, which allows the separation of ferrous and ferric complexes in 2 min.