The in vivo and in vitro determination of significant intracellular stavudine (d4T) triphosphate (d4TTP) concentrations in human immunodeficiency virus (HIV)-infected subjects and NS-1 cells treated with zidovudine (ZDV) has recently been reported. This study was conducted to corroborate these findings with in vivo samples from HIV-infected subjects taking ZDV and in vitro CEM SS cells incubated with different ZDV concentrations. Previously, we have reported on our validated high-performance liquid chromatography coupled with tandem mass spectrometry methodology for the simultaneous determination of d4TTP, lamivudine triphosphate, and ZDV triphosphate (ZDVTP) concentrations. Using this methodology, we monitored the d4TTP concentration in more than 100 samples from HIV-infected subjects treated with d4T. In addition, we simultaneously monitored the concentrations of d4TTP and ZDVTP in more than 500 samples from HIVinfected individuals who were taking ZDV. Finally, we performed in vitro studies by incubating CEM SS cells with 10 M, 50 M, and 100 M ZDV and monitored the formation of d4TTP at 24 and 48 h. We could measure d4TTP concentrations from HIV-infected individuals with a limit of quantitation (LOQ) of 2.7 fmol/10 6 cells (total injection, 54 fmol). In the in vivo studies, we measured the d4TTP concentrations among patients receiving d4T treatment, but the samples from patients taking ZDV did not provide d4TTP concentrations above the LOQ. Furthermore, in vitro samples did not produce any signal for d4TTP, despite the detection of substantial ZDVTP concentrations in CEM SS cells. Thus, contrary to the previous report, we found no evidence for the in vivo or in vitro transformation of ZDVTP to d4TTP in HIV-infected subjects or CEM SS cells.Nucleoside reverse transcriptase inhibitors are an essential part of the highly active antiretroviral therapy implemented for the treatment of human immunodeficiency virus (HIV)-infected individuals (24). Two of the most widely used nucleoside reverse transcriptase inhibitors are zidovudine (ZDV) (8) and stavudine (d4T) (7). ZDV and d4T are thymidine nucleoside analogs that need to be converted into their active forms, zidovudine triphosphate (ZDVTP) and stavudine triphosphate (d4TTP), to be effective against HIV replication (6, 14). Furthermore, it has been established that ZDV and d4T use the same intracellular mechanisms for their activation processes (6, 14), and combination therapy with these two analogs is not recommended (12,15,16).Various methods (indirect and direct detection of nucleotides) have been developed for the determination of intracellular ZDVTP and d4TTP in vivo. The indirect methodologies fractionate the phosphate anabolites (ZDV monophosphate, ZDV diphosphate, ZDVTP, d4T monophosphate, d4T diphosphate, and d4TTP) by the use of ion-exchange cartridges or ionic liquid chromatography, followed by dephosphorylation and quantification of the parent drug (ZDV and d4T) by radioimmunoassays or by high-performance liquid chromatography coupled with tandem mass s...

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