Direct determination of phosphorylated intracellular anabolites of stavudine (d4T) by liquid chromatography tandem mass spectrometry

The objective was to develop and validate a routine assay for active intrac ellular anabolites of stavudine (d4T), a nucleoside reverse transcriptase i nhibitor in human PBMC, applicable to pharmacokinetic studies and treatment monitoring. This was achieved using liquid chromatography coupled to tande m mass spectrometry (LC/MS/MS), which theoretically allies optimum sensitiv ity, specificity and high sample throughput. After cellular lysis in a Tris /methanol buffer, the extract spiked with (2)[H-8]-ATP (internal standard) is directly injected into the LC/MS/MS system. Phosphorylated metabolites o f d4T as well as deoxythymidine-triphosphate, the competitor on the reverse transcriptase, are separated from d4T on a reverse-phase microbore column with ion pairing. The detection is performed in the multiple reaction monit oring (MRM) mode after drug ionisation in negative mode electrospray. The l imit of quantitation for d4T-TP was 138 fmol per 7 mL blood (9.8 fmol per 1 0(6) cells) and CV% for repeatability and intermediate precision were lower than 15%. Stability of compounds was checked before and during the process of isolation of PBMC. Cellular samples from several d4T-treated patients w ere successfully analysed using this method and d4T-triphosphate and deoxyt hymidine triphosphate were recovered. In conclusion, we have developed and validated a routine LC/MS/MS method that allows the simultaneous determinat ion of mono-, di-and triphosphorylated anabolites of d4T in PBMC as well as the natural corresponding triphosphate in one analysis. For the first time , the chain terminator ratio (d4T-TP/dT-TP) could be directly measured. Thi s method can be used simply and routinely on more than 35 samples per day. Extension to other nucleoside analogues is under development. Copyright (C) 2001 John Wiley & Sons, Ltd.