Determination of ferric iron chelators by high-performance liquid chromatography using luminol chemiluminescence detection

• A novel HPLC system was devised to analyze ferric ion chelators in higher plants.
• Detection of ferric ion chelators was based on a luminol/H2O2 system.
• The method can detect minute levels of ferric ion chelators.

Iron is an essential element for higher plants, and its acquisition and transportation is one of the greatest limiting factors for plant growth because of its low solubility in normal soil pHs. Higher plants biosynthesize ferric iron [Fe(III)] chelator (FIC), which solubilizes the iron and transports it to the rhizosphere. A high-performance liquid chromatography (HPLC) post-column method has been developed for the analysis of FICs using the luminol/H2O2 system for chemiluminescence (CL) detection. A size-exclusion column was the most suited in terms of column efficiency and CL detection efficiency. Mixing of the luminol with H2O2 in a post-column reaction was feasible, and a two-pump system was used to separately deliver the luminol and H2O2 solutions. The luminol and H2O2 concentrations were optimized using Fe(III)–EDTA and Fe(III)–citrate (Cit) solutions as analytes. A strong CL intensity was obtained for Fe(III)-Cit when EDTA was added to the luminol solution, probably because of an exchange of Cit with EDTA after separation on the HPLC column; CL efficiency was much higher for Fe(III)–EDTA than for Fe(III)–Cit with the luminol/H2O2 system. The present method can detect minute levels of Fe(III)–FICs; the detection limits of Fe(III)–EDTA, Fe(III)–Cit and Fe(III)–nicotianamine were 0.77, 2.3 and 1.1 pmol, respectively.