A gradient ion-pair HPLC separation of highly hydrophilic 3-hydroxypyridin-4-one (HPO) iron chelators is described. The separation of HPOs was performed using a reversed-phase polymer HPLC column (PLRP-S 100 A, 15x0.46 cm ID, 5 microm). The ion-pair buffer contained 1-heptanesulfonic acid (sodium salt) (5 mM) and the pH was adjusted to 2.0 using HCl. The gradient was 2%-35% CH3CN in 20 min and post-run was followed for 5 min using 2% CH3CN and 98% buffer. The flow-rate was 1 ml/min and the analytes were monitored at 280 nm. The retention times of 30 hydrophilic HPOs fell in the range of 10-18 min with sharp peak shapes, although these iron chelators possess various functional groups and distribution coefficients. The application of this HPLC method in the analysis of HPO chelators and their metabolites in rat bile and urine is described.