High-performance liquid chromatography method for ferric iron chelators using a post-column reaction with Calcein Blue

• An HPLC system was devised to detect and quantitate ferric iron chelators.
• This system takes advantage of the chelators’ ability to bind Fe(III) ions.
• This strategy allows detection of ferric iron chelators alone.
• The method was applied to ferric ion chelators in the xylem sap of barley.

Iron (Fe) is an essential element for higher plants, which take it up from the soil at the root surface and transport it to shoots through the xylem. Fe(III) chelators, such as organic acids and phytosiderophores, play important roles in the acquisition and transportation of Fe(III). Therefore, a selective and sensitive method for analyzing Fe(III) chelators is required to study the many Fe-related physiological mechanisms in plants. A novel analytical approach employing a high-performance liquid chromatography post-column method with fluorescence detection was developed to separate and detect Fe(III) chelators. This method takes advantage of the quenching of the fluorescence of Calcein Blue (CB) that occurs with the formation of an Fe(III)–CB complex and the dequenching that occurs with the release of CB as a result of competition for Fe(III) between CB and an Fe(III) chelator. This simple experimental method does not require complicated pretreatments and can selectively detect Fe(III) chelators according to their Fe(III)-chelating ability. The detection limit for citric acid using this method was 72 pmol. Furthermore, this method can also detect unknown Fe(III) chelators that exhibit a high affinity for Fe(III). The method was evaluated with xylem sap of barley, which was shown to contain several Fe(III) chelators.