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A new high-performance liquid chromatography method was developed to study the diffusion of halofuginone in the wall of the ureter. The human ureter extracts were prepared by trypsin digestion of the tissues followed by liquid–liquid extraction using the isopropanol after precipitating the proteins. The method used a reversed-phase C18 column with a mobile phase delivered to the analytical column according to a gradient program starting at a composition of ammonium acetate (pH 4.7; 10 mM)-acetonitrile-triethylamine (70:30:0.2, v/v/v) and linear changes to 90% of acetonitrile at 11 min. Liquid-liquid extraction proved to be selective for the HFG and provided a high recovery rate of 97.7%. The HPLC method was successfully validated by applying the novel validation protocol using the accuracy profile based on a new concept, that of the total error. The protocol V4, with five levels of concentration and 105 trials, was selected according to the algorithm designed by the SFSTP 2003 committee. Acceptance limits were set up at 20%, while the risk was settled at 5%. The method was found accurate over a concentration range of 0.2–10 μg/ml. The limit of detection for HFG was 0.06061 μg/ml. In order to demonstrate the applicability of this method in ex vivo application, the quantification of HFG in the wall was applied to study its distribution from a gel (0.03% w/w of HFG) in the human ureter
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