Simultaneous quantitation of the nucleotide analog adefovir, its phosphorylated anabolites and 2′-deoxyadenosine triphosphate by ion-pairing LC/MS/MS

The nucleotide analog adefovir is an important therapy for hepatitis B viral infection. The study of nucleoside/tide pharmacology has been hampered by difficulties encountered when trying to develop LC/MS/MS methods for these polar analytes. In an attempt to identify a more convenient, selective and sensitive alternative to the analysis of the metabolism of radiolabled parent nucleotide traditionally used for in vitro cell culture studies, an LC/MS/MS method was developed for the quantitative detection of adefovir and its phosphorylated metabolites in cellular samples. Ion-pairing reversed phase LC using tetrabutylammonium (TBA) and ammonium phosphate had the best compromise between chromatographic separation and positive mode MS/MS detection. Using microbore reverse phase columns and a low flow acetonitrile gradient it was possible to quantitate adefovir, its metabolites and 2′-deoxyadenosine triphosphate. A cross-validation showed comparable levels of adefovir and its metabolites were determined using either LC/MS/MS or radioactivity detection. However, initial methods were conducted at high pH and utilized an acetonitrile step gradient causing unacceptable column life and unpredictable equilibration. Further method optimization lowered the concentration of TBA and phosphate, decreased pH and applied a linear gradient of acetonitrile. This work resulted in a method that was found to have sensitivity, accuracy and precision sufficient to be a useful tool in the study of the intracellular pharmacology of adefovir in vitro and may be more broadly applicable.