Abstract
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 0-25 mM 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 (100 microL 10% formic acid in H(2)O, v/v; 2.65 M) 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.