Becher François, Schlemmer Dimitri, Pruvost Alain, Nevers Marie-Claire, Goujard Cécile, Jorajuria Sylvie, Guerreiro Catherine, Brossette Thierry, Lebeau Luc, Créminon Christophe, Grassi Jacques, Benech Henri
Pharmacology and Immunology Unit, CEA, DSV/DRM, CEA/Saclay, Gif-sur-Yvette, France.
Anal Chem. 2002 Aug 15;74(16):4220-7. doi: 10.1021/ac020144r.
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.
最近已开发出直接液相色谱/串联质谱法（Direct LC/MS/MS），用于测量HIV阳性患者外周血单核细胞（PBMCs）中几种核苷类逆转录酶抑制剂（NRTI）的三磷酸代谢物。虽然齐多夫定（AZT）是最常用的NRTIs之一，但其活性代谢物AZT-TP尚未开发出此类方法，主要是因为存在干扰此类检测的内源性核苷酸。在本文中，我们首先描述了PBMCs中AZT-TP的两种酶免疫测定法（EIA）的开发：一种直接测量AZT-TP含量；另一种是在选择性提取AZT-TP并去磷酸化后测量核苷AZT。这两种测定法的精密度太低，无法精确测定PBMC样品中的AZT-TP。直接LC/MS/MS对AZT-TP的特异性不足，因为在PBMCs的细胞内介质中发现至少两种干扰性内源性核苷酸（与AZT-TP具有相同的质荷比、片段以及保留时间相近）。免疫亲和萃取（IAE）与LC/MS/MS的离线联用被证明是一种成功的策略，无需去磷酸化就能实现适当的特异性和灵敏度（定量限设定为9.3 fmol/10(6) 个细胞），以测定来自7 mL HIV感染患者血液的PBMCs中的AZT-TP。这种IAE-LC/MS/MS方法的验证表明，重复性和中间精密度的变异系数百分比低于15%。一名分析人员每周可分析超过150个样品，使该方法适用于临床研究期间的常规分析。
