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

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