Analysis of intracellular didanosine triphosphate at sub-ppb level using LC-MS/MS

An analytical procedure has been developed for the analysis of intracellular didanosine triphosphate (ddATP). An electrospray ionization tandem mass spectrometer (ESI-MS) was interfaced to liquid chromatography (LC) using a mobile phase CH3OH/H2O (25/75) containing 1% formic acid for the analysis of the 5'-triphosphate metabolite of the antiviral didanosine. In this procedure, ddATP was extracted from CEM-T4 cells, isolated using an exchange anion solid phase extraction procedure, enzymatically dephosphorylated and then analyzed by LC-MS/MS within a 1 min run time. The influence of several parameters (electrospray ionization interface, acidic modifiers of the mobile phase) has been studied. A calibration curve was generated and the linear regression analysis yielded a regression coefficient (r(2)) greater than 0.999. Using LC-MS/MS detection in single reaction monitoring mode (SRM), the limit of quantitation of ddA in CEM-T4 cells was 0.02 ng ml(-1). Furthermore, this procedure could be used to perform simultaneous detection of five nucleoside reverse transcriptase inhibitors, such as AZT, 3TC, ddA, ddC and d4T and make LC-MS/MS a method of choice for Therapeutic Drug Monitoring (TDM) in a clinical environment.