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A strategy for liquid chromatography/tandem mass spectrometric assays of intracellular drugs: application to the validation of the triphosphorylated anabolite of antiretrovirals in peripheral blood mononuclear cells
Author(s) -
Becher François,
Pruvost Alain,
Gale Jane,
Couerbe Philippe,
Goujard Cécile,
Boutet Valerie,
Ezan Eric,
Grassi Jacques,
Benech Henri
Publication year - 2003
Publication title -
journal of mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 1076-5174
DOI - 10.1002/jms.500
Subject(s) - chemistry , intracellular , peripheral blood mononuclear cell , analyte , nucleoside , chromatography , liquid chromatography–mass spectrometry , reverse transcriptase inhibitor , tandem mass spectrometry , lysis , didanosine , stavudine , pharmacology , biochemistry , mass spectrometry , zidovudine , reverse transcriptase , human immunodeficiency virus (hiv) , virology , in vitro , biology , rna , gene , viral disease
The pharmacokinetics of intracellular drugs have recently aroused new interest because monitoring a drug's behaviour near the site of action can enhance knowledge of its efficacy and toxicity. Liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) is particularly attractive for intracellular analytes. Very few papers deal precisely with special features encountered in intracellular drug assay or with how closely the assay matches the actual recommendations. Particular problems are encountered mainly because the analytes are located intracellularly. This mainly concerns the handling of biological media, including provision of blank samples using Ficoll gradient separation, cell counts, optimisation of cell lysis, sample extraction, plotting standard curves using either fmol/10 6 cells or fmol/ml of extract or fmol/sample, the matrix effect as a function of the number of cells, stability before and during cell separation, as well as in storage conditions using clinical samples, biological matrix replacement and interference by endogenous compounds. This paper describes a strategy for the full validation and routine use of an LC/MS/MS assay applied to the simultaneous intracellular determination of the triphosphorylated anabolites of didanosine (2′,3′‐dideoxyadenosine triphosphate or ddA‐TP) and stavudine (2′,3′‐didehydro‐3′‐deoxythymidine triphosphate or d4T‐TP), two nucleoside reverse transcriptase inhibitors of HIV, in human peripheral blood mononuclear cells (PBMCs), as a guide for further LC/MS/MS assay of intracellular drugs. Copyright © 2003 John Wiley & Sons, Ltd.