Analysis of ⁵⁵Fe-labeled hydroxamate siderophores by high-performance liquid chromatography

The radiolabeled iron(III) chelates of four different hydroxamate siderophores (desferrioxamine B, rhodotorulic acid, desferriferrichrome and desferriferrichrome A) were analyzed by high-performance liquid chromatography using an absorbance or radioactivity detector. Optimal conditions consisted of a polymeric PRP-1 column and a water to acidified acetonitrile gradient as mobile phase. Silica based columns gave similar resolution, but adsorption of unchelated iron-55 to the silica support limited the desirability of this approach. Detection limits based on radioactivity of the labeled compounds were consistently two to three orders of magnitude greater than that observed using visible absorbance at 436 nm. This method provides a generalized approach for the detection of high affinity iron(III) chelators (e.g. siderophores), regardless of their structural type.