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Quantitative determination of labile iron complexes by hydrophilic interaction liquid chromatography – electrospray MS/inductively coupled plasma mass spectrometry


Iron is an important essential element for life. While the essentiality of iron (Fe) for plant growth was established by E. Gris as early as 1843, it is more recently that research towards the understanding of the mechanisms responsible for its absorption, transport and assimilation in staple plants has been stimulated. Whereas several iron complexes have been identified by methods of speciation analysis such as HPLC-electrospray MS/MS, their lability limits the quantitative determination after chromatographic separation. The main problem is the presence of several labile metal complexes in the state of fragile equilibrium that is easily disturbed by interaction with the chromatographic stationary phase or changing sample pH.  Also compound independent iron response along the chromatographic separation is often compromised by changes in the eluent composition during gradient elution.

The new study:
These difficulties motivated the group of researchers working in Pau to develop a method for quantitative iron speciation analysis in plant liquids by adding isotopically labelled Fe(III) and letting it react with the bioligands present to reproduce the original molecular speciation of iron.  In this way, species-specific internal standards can be produced for each iron species present in the sample, without the need for the preliminary knowledge of their identity. Following the separation of iron species by hydrophilic interaction ion chromatography (HILIC), each species can then be quantified on the basis of the ratio of the naturally present and isotopically enriched added iron by either measuring the iron isotopes by ICP-MS or the iron-containing species by ESI-MS.  This species-specific isotope dilution approach is able to correct for on-column phenomena, such as species retention or degradation as well as sensitivity changes during gradient elution or signal drift.

Using coconut water as an example of endosperm liquid, the approach was applied to the quantification of mixed ligand multicore iron complexes with malate and citrate. Samples were spiked with 57Fe one day before analysis in order to allow for sufficient time for complex formation. The authors admit some constraints: The two isotopes must be interference free and for HILIC-ICP-MS the species must be baseline separated. Also the consumption of the 57Fe spike by exchanging with the originally present 56Fe resulting in the formation of nixed isotopic species must be corrected. The authors concluded, that the method can be extended to labile complexes of other metals, if these constraints are well handled.

The original study

Katarzyna Kinska, Ghaya Alchoubassi, Luluil Maknun, Katarzyna Bierla, Ryszard Lobinski, Joanna Szpunar, Quantitative determination of iron citrate/malate complexes by isotope–dilution hydrophilic interaction liquid chromatography – electrospray MS/inductively coupled plasma MS, J. Anal. At. Spectrom., 2022. DOI: 10.1039/d2ja00164k

Instrumentation used:

Dionex Ultimate 3000 RS HPLC system
Agilent Technologies - 7700s ICP-MS
Thermo Scientific - Q Exactive Plus Quadrupole-Orbitrap Hybrid Mass Spectrometer

Related studies (newest first)

G. Alchoubassi, K. Kinska, K. Bierla, R. Lobinski, J. Szpunar, Speciation of essential nutrient trace elements in coconut water, Food Chem., 2021, 339. DOI: 10.1016/j.foodchem.2020.127680

H. N. Brawley and P. A. Lindahl, Low-molecular-mass labile metal pools in Escherichia coli: advances using chromatography and mass spectrometry, J. Biol. Inorg. Chem., 26 (2021) 479–494. DOI: 10.1007/s00775-021-01864-w

T. Q. Nguyen, J. E. Kim, H. N. Brawley, P. A. Lindahl, Chromatographic detection of low-molecular-mass metal complexes in the cytosol of Saccharomyces cerevisiae, Metallomics, 12 (2020) 1094–1105. DOI: 10.1039/c9mt00312f

P. Flis, L. Ouerdane, L. Grillet, C. Curie, S. Mari, R. Lobinski, Inventory of metal complexes circulating in plant fluids: a reliable method based on HPLC coupled with dual elemental and high-resolution molecular mass spectrometric detection, New Phytol., 211 (2016) 1129–1141. DOI: 10.1111/nph.13964

L. Grillet, L. Ouerdane, P. Flis, M. T. T. Hoang, M. P. Isaure, R. Lobinski, C. Curie and S. Mari, Ascorbate Efflux as a New Strategy for Iron Reduction and Transport in Plants, J. Biol. Chem., 289/5 (2014) 2515–2525. DOI: 10.1074/jbc.M113.514828

A. Alvarez-Fernández, P. Díaz-Benito, A. Abadía, A.F. López Millán, J. Abadía, Metal species involved in long distance metal transport in plants, Front. Plant Sci., 2014, 5, 105. DOI: 10.3389/fpls.2014.00105

J. Köster, Rongli Shi, N. von Wirén, G. Weber, Evaluation of different column types for the hydrophilic interaction chromatographic separation of iron-citrate and copper-histidine species from plants, J. Chromatogr. A, 1218/30 (2011) 4934-4943. DOI: 10.1016/j.chroma.2011.03.036

R. Rellán-Alvarez, J. Giner-Martínez-Sierra, J. Orduna, I. Orera, J. A. Rodríıguez-Castrillón, J.I. García-Alonso, J. Abadía, A. Alvarez-Fernández, Identification of a Tri-Iron(III), Tri-Citrate Complex in the Xylem Sap of Iron-Defi cient Tomato Resupplied with Iron: New Insights into Plant Iron Long-Distance Transport, Plant Cell Physiol., 51 (2009) 91–102. DOI: 10.1093/pcp/pcp170

Isabelle Gautier-Luneau, Claire Merle, Delphine Phanon, Colette Lebrun, Frédéric Biaso, Guy Serratrice, Jean-Louis Pierre, New Trends in the Chemistry of Iron(iii) Citrate Complexes: Correlationsbetween X-ray Structures and Solution Species Probed by Electrospray Mass Spectrometry and Kinetics of Iron Uptake from Citrate by Iron Chelators, Chem. Eur. J., 11 (2005) 2207-2219. DOI: 10.1002/chem.200401087

last time modified: September 13, 2022


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