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

摘要

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_3CN) and the retention of the anionic nucleotides with an eluting ion (bicarbonate). Separation of all analytes was achieved using a 0-25 mM ammonium bicar_bonate gradient in CH_3CN-H_2O (15:85, vJv). Under these conditions, however, very long re-equilibration times were required. Injection of an acidic solution (100 _L 10% formic acid in H_2O, 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_2O_2) solution (0.05% H_20_2 in CH_3CN-H_20 (15:85, v/v), pH 4) before starting an analytical run. The oxidation of the stationary phase with H_20_2 prevented its slow reduction, which most likely caused the decreasing retention times. The analytes were detected using tandem mass spectrometry.