Retention studies of 2′-2′-difluorodeoxycytidine and 2′-2′-difluorodeoxyuridine nucleosides and nucleotides on porous graphitic carbon: Development of a liquid chromatography–tandem mass spectrometry method

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, CH3CN) 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 CH3CN–H2O (15:85, v/v). Under these conditions, however, very long re-equilibration times were required. Injection of an acidic solution (100 μL 10% formic acid in H2O, 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 (H2O2) solution (0.05% H2O2 in CH3CN–H2O (15:85, v/v), pH 4) before starting an analytical run. The oxidation of the stationary phase with H2O2 prevented its slow reduction, which most likely caused the decreasing retention times. The analytes were detected using tandem mass spectrometry.