Development of a direct assay for measuring intracellular AZT triphosphate in humans peripheral blood mononuclear cells
| Autor: | Dimitri Schlemmer, Henri Benech, Catherine Guerreiro, Sylvie Jorajuria, Luc Lebeau, Cécile Goujard, Thierry Brossette, Christophe Créminon, Jacques Grassi, François Becher, Alain Pruvost, Marie-Claire Nevers |
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| Přispěvatelé: | Service de Pharmacologie et d'Immunologie, Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de Biologie et de Technologies de Saclay (IBITECS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, Hôpital Bicêtre, Université Paris-Sud - Paris 11 (UP11)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Bicêtre, CEA [Fontenay-aux-Roses] (UGRA / SETA), Chimie Organique, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Université Louis Pasteur - Strasbourg I, Conception et application de molécules bioactives (CAMB), Université de Strasbourg (UNISTRA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Service de Pharmacologie et Immunoanalyse (SPI), Médicaments et Technologies pour la Santé (MTS), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut de biologie et de technologies de Saclay, Université Paris-Saclay-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Hôpital Bicêtre-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Université Paris-Sud - Paris 11 (UP11), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS) |
| Rok vydání: | 2002 |
| Předmět: |
Anti-HIV Agents
[SDV]Life Sciences [q-bio] 01 natural sciences High-performance liquid chromatography Peripheral blood mononuclear cell Mass Spectrometry Analytical Chemistry Dephosphorylation Immunoenzyme Techniques 03 medical and health sciences Zidovudine immune system diseases medicine [CHIM]Chemical Sciences Humans Thymine Nucleotides Nucleotide MESH: Anti-HIV Agents MESH: Chromatography High Pressure Liquid MESH: Immunoenzyme Techniques Chromatography High Pressure Liquid MESH: Mass Spectrometry chemistry.chemical_classification MESH: Dideoxynucleotides 0303 health sciences MESH: Humans Chromatography Reverse-transcriptase inhibitor 030306 microbiology 010401 analytical chemistry MESH: Zidovudine virus diseases Reproducibility of Results biochemical phenomena metabolism and nutrition MESH: Thymine Nucleotides 0104 chemical sciences MESH: Reproducibility of Results MESH: Leukocytes Mononuclear chemistry Leukocytes Mononuclear Nucleoside Quantitative analysis (chemistry) medicine.drug Dideoxynucleotides |
| Zdroj: | Analytical Chemistry Analytical Chemistry, 2002, 74 (16), pp.4220-4227. ⟨10.1021/ac020144r⟩ Analytical Chemistry, American Chemical Society, 2002, 74 (16), pp.4220-4227. ⟨10.1021/ac020144r⟩ |
| ISSN: | 0003-2700 1520-6882 |
| DOI: | 10.1021/ac020144r⟩ |
| Popis: | International audience; 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. |
| Databáze: | OpenAIRE |
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