title: Direct determination of phosphorylated intracellular anabolites of stavudine (d4T) by liquid chromatography/tandem mass spectrometry.
abstract: The objective was to develop and validate a routine assay for active intracellular anabolites of stavudine (d4T), a nucleoside reverse transcriptase inhibitor in human PBMC, applicable to pharmacokinetic studies and treatment monitoring. This was achieved using liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS), which theoretically allies optimum sensitivity, 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 of 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 monitoring (MRM) mode after drug ionisation in negative mode electrospray. The limit of quantitation for d4T-TP was 138 fmol per 7 mL blood (9.8 fmol per 10(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 were successfully analysed using this method and d4T-triphosphate and deoxythymidine triphosphate were recovered. In conclusion, we have developed and validated a routine LC/MS/MS method that allows the simultaneous determination 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. This method can be used simply and routinely on more than 35 samples per day. Extension to other nucleoside analogues is under development.
authors.affiliation: CEA, Pharmacology and Immunology Unit, DSV/DRM, CEA/Saclay, 91191 Gif-sur-Yvette cedex, France.
authors.forename: A
authors.initials: A
authors.lastname: Pruvost
authors.forename: F
authors.initials: F
authors.lastname: Becher
authors.forename: P
authors.initials: P
authors.lastname: Bardouille
authors.forename: C
authors.initials: C
authors.lastname: Guerrero
authors.forename: C
authors.initials: C
authors.lastname: Creminon
authors.forename: J F
authors.initials: JF
authors.lastname: Delfraissy
authors.forename: C
authors.initials: C
authors.lastname: Goujard
authors.forename: J
authors.initials: J
authors.lastname: Grassi
authors.forename: H
authors.initials: H
authors.lastname: Benech
mesh_terms: D019380:Anti-HIV Agents / Q000097:blood / Q000493:pharmacokinetics; D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods; D003854:Deoxyribonucleotides / Q000097:blood; D016903:Drug Monitoring / Q000379:methods; D006801:Humans; D007202:Indicators and Reagents; D008214:Lymphocytes / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D010766:Phosphorylation; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D018119:Stavudine / Q000031:analogs & derivatives* / Q000097:blood / Q000493:pharmacokinetics*
chemical_list: D019380:Anti-HIV Agents; D003854:Deoxyribonucleotides; D007202:Indicators and Reagents; D018119:Stavudine