Quantitative analysis of gemcitabine triphosphate in human peripheral blood mononuclear cells using weak anion-exchange liquid chromatography coupled with tandem mass spectrometry

Gemcitabine triphosphate (dFdCTP) is a highly active metabolite of gemcitabine. It is formed intra-cellularly via the phosphorylation of gemcitabine by deoxycytidine kinase. The monitoring of dFdCTP in human peripheral blood mononuclear cells (PBMCs), in addition to plasma concentrations of gemcitabine and its metabolite 2',2'-difluorodeoxyuridine, is considered very useful in determining pharmacokinetic-pharmacodynamic relationships.We describe a novel sensitive assay for the quantification of dFdCTP in human PBMCs. The method is based on weak anion-exchange liquid chromatography and detection with tandem mass spectrometry (LC-MS/MS). The assay has been validated from 1 ng/ml (lower limit of quantification, LLOQ) to 25 ng/ml (upper limit of quantification, ULOQ) using 180 microl aliquots of PBMC extracts containing approximately 0.648 mg protein or 3.8 x 10(6) lysed PBMCs. The LLOQ is equivalent to 94 fmol/10(6) cells (1 ng/ml = 0.18 ng/180 microl or 0.18 ng/0.648 mg protein = 0.047 ng/10(6) cells or 94 fmol/10(6) cells). This highly sensitive assay is capable of quantifying about 200-fold lower concentrations of dFdCTP in human PBMCs than currently available methods.