An analytical procedure has been developed for the analysis of intracellula
r didanosine triphosphate (ddATP). An electrospray ionization tandem mass s
pectrometer (ESI-MS) was interfaced to liquid chromatography (LC) using a m
obile phase CH3OH/H2O (25/75) containing 1% formic acid for the analysis of
the 5'-triphosphate metabolite of the antiviral didanosine. In this proced
ure. ddATP was extracted from CEM-T4 cells, isolated using an exchange anio
n solid phase extraction procedure, enzymatically dephosphorylated and then
analyzed by LC-MS/MS within a I min run time. The influence of several par
ameters (electrospray ionization interface, acidic modifiers of the mobile
phase) has been studied. A calibration curve was generated and the linear r
egression analysis yielded a regression coefficient (r(2)) greater than 0.9
99. 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). Furthermor
e, this procedure could be used to perform simultaneous detection of five n
ucleoside reverse transcriptase inhibitors, such as AZT, 3TC. ddA, ddC and
d4T and make LC-MS/MS a method of choice for Therapeutic Drug Monitoring (T
DM) in a clinical environment. (C) 2001 Elsevier Science B.V. All rights re
served.