The development of a method for the separation of 2′-2′-difluorodeoxycytidine (gemcitabine, dFdC), 2′-2′-difluorodeoxyuridine (dFdU) and their mono-, di- and triphosphates using a porous graphitic carbon column (Hypercarb), without ion-pairing agent, is described. The retention of dFdC and dFdU could be controlled with an organic modifier (acetonitrile, CH 3 CN) and the retention of the anionic nucleotides with an eluting ion (bicarbonate). Separation of all analytes was achieved using a 0–25mM ammonium bicarbonate gradient in CH 3 CN–H 2 O (15:85, v/v). Under these conditions, however, very long re-equilibration times were required. Injection of an acidic solution (100μL 10% formic acid in H 2 O, v/v; 2.65M) after running a gradient directly restored the separation capabilities of the column. Still, separation between the analytes slowly deteriorated over a period of months. These problems were solved by preconditioning the column with a pH buffered hydrogen peroxide (H 2 O 2 ) solution (0.05% H 2 O 2 in CH 3 CN–H 2 O (15:85, v/v), pH 4) before starting an analytical run. The oxidation of the stationary phase with H 2 O 2 prevented its slow reduction, which most likely caused the decreasing retention times. The analytes were detected using tandem mass spectrometry.