Reversed-phase high-performance liquid chromatography of non-transferrin-bound iron and some hydroxypyridone and hydroxypyrone chelators.

The pursuit of orally available Fe(III) chelating agents has resulted in several clinical trials of 1,2-dimethyl-3-hydroxypyrid-4-one (CP20). Chromatography of this and related Fe chelators on silica-based columns has proven difficult due to unwanted interactions with the stationary phase, including with contaminating Fe bound to silanol groups. By addition of Fe3+ (50 microM ferric ammonium citrate) to an acidified aqueous mobile phase, we have successfully separated a series of hydroxypyridones-including CP20-and the related pyrones maltol and ethylmaltol by HPLC on microBondapak C18. Complexation occurs with these agents even at low pH, and they elute in an order consistent with the partition coefficients of their Fe(III) complexes. By the reverse strategy of adding ethylmaltol to the mobile phase, chelatable Fe was chromatographed and the peak response at 500 nm was linear down to a detection limit below 0.5 microM. This method was applied to pooled serum and to serum spiked with Fe after filtration at 10 kDa cut-off. The direct determination of non-transferrin-bound Fe at micromolar concentrations in serum is possible with this approach.