Chromatographic methods for the separation of biocompatible iron chelators from their synthetic precursors and iron chelates

Chromatographic methods have been developed for the separation of the three novel biocompatible iron chelators pyridoxal isonicotinoyl hydrazone (PIH), salicylaldehyde isonicotinoyl hydrazone (SIH), and pyridoxal 2-chlorobenzoyl hydrazone (o-108) from their synthetic precursors and iron chelates. The chromatographic analyses were achieved using analytical columns packed with 5 microm Nucleosil 120-5 C18. For the evaluation of all chelators in the presence of the synthetic precursors, EDTA was added to the mobile phase at a concentration of 2 mM. The best separation of PIH and its synthetic precursors was achieved using a mixture of phosphate buffer (0.01 M NaH2PO4, 5 mM 1-heptanesulfonic acid sodium salt; pH 3.0) and methanol (55:45, v/v). For separation of SIH and its synthetic precursors, the mobile phase was composed of 0.01 M phosphate buffer (pH 6.0) and methanol (60:40, v/v). o-108 was analyzed employing a mixture of 0.01 M phosphate buffer (pH 7.0), methanol, and acetonitrile (60:20:20, v/v/v). These mobile phases were slightly modified to separate each chelator from its iron chelate. Furthermore, a RP-TLC method has also been developed for fast separation of all compounds. The chromatographic methods described herein could be applied in the evaluation of purity and stability of these drug candidates.