Development of a direct assay for measuring intracellular AZT triphosphate in humans peripheral blood mononuclear...

Direct LC/MS/MS methods have recently been developed for measuring triphosphate anabolites of several nucleosidic reverse transcriptase inhibitor (NRTI) in peripheral blood mononuclear cells (PBMCs) from HIV-positive patients. Whereas AZT is one of the most-used NRTIs, no such method has been developed for AZT-TP, its active anabolite, mainly because of the presence of endogenous nucleotides that interfere with such an assay. In this paper, we first describe the development of two enzyme immunoassays (EIA) of AZT-TP in PBMCs: one directly measuring AZT-TP content; the other, measuring the nucleoside AZT after selective extraction of AZT-TP and dephosphorylation. The precision of these two assays was too low to achieve precise determination of AZT-TP in PBMC samples. Direct LC/MS/MS is not specific enough for AZT-TP, since at least two interfering endogenous nucleotides (same m/z ratio and fragment as well as retention time close to that of AZT-TP) are found in the intracellular medium of PBMCs. The off-line combination of immunoaffinity extraction (IAE) and LC/MS/MS proved to be a successful strategy allowing without dephosphorylation appropriate specificity and sensitivity (limit of quantification established as 9.3 fmol/10(6) cells) to determine AZT-TP in PBMCs from 7 mL of blood of HIV-infected patients. Validation of this IAE-LC/MS/MS method demonstrated CV percent for repeatability and intermediate precision lower than 15%. More than 150 samples/week can be analyzed by one analyst, making this method suitable for routine analysis during clinical studies.